AU2003279430B2 - Method of determining specific groups forming heparins - Google Patents
Method of determining specific groups forming heparins Download PDFInfo
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- AU2003279430B2 AU2003279430B2 AU2003279430A AU2003279430A AU2003279430B2 AU 2003279430 B2 AU2003279430 B2 AU 2003279430B2 AU 2003279430 A AU2003279430 A AU 2003279430A AU 2003279430 A AU2003279430 A AU 2003279430A AU 2003279430 B2 AU2003279430 B2 AU 2003279430B2
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- deoxy
- anhydro
- acid
- heparins
- heparinase
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- 238000000034 method Methods 0.000 title claims description 35
- 229960002897 heparin Drugs 0.000 title claims description 15
- 229920000669 heparin Polymers 0.000 title claims description 15
- 108010022901 Heparin Lyase Proteins 0.000 claims description 29
- 150000002016 disaccharides Chemical class 0.000 claims description 25
- 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 18
- 150000002482 oligosaccharides Polymers 0.000 claims description 17
- 239000011734 sodium Substances 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 13
- 229940127215 low-molecular weight heparin Drugs 0.000 claims description 11
- 238000001514 detection method Methods 0.000 claims description 10
- 150000001720 carbohydrates Chemical class 0.000 claims description 7
- 238000005571 anion exchange chromatography Methods 0.000 claims description 5
- 235000000346 sugar Nutrition 0.000 claims description 5
- 150000008163 sugars Chemical class 0.000 claims description 5
- 238000003556 assay Methods 0.000 claims description 4
- 150000004043 trisaccharides Chemical class 0.000 claims description 4
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical class CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 3
- -1 alkali metal salt Chemical class 0.000 claims description 3
- 239000003638 chemical reducing agent Substances 0.000 claims description 3
- 238000004587 chromatography analysis Methods 0.000 claims description 3
- 238000012691 depolymerization reaction Methods 0.000 claims description 3
- BAZAXWOYCMUHIX-UHFFFAOYSA-M sodium perchlorate Chemical compound [Na+].[O-]Cl(=O)(=O)=O BAZAXWOYCMUHIX-UHFFFAOYSA-M 0.000 claims description 3
- 229910001488 sodium perchlorate Inorganic materials 0.000 claims description 3
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 2
- 229910052783 alkali metal Inorganic materials 0.000 claims 1
- 150000001450 anions Chemical class 0.000 claims 1
- 125000002467 phosphate group Chemical class [H]OP(=O)(O[H])O[*] 0.000 claims 1
- 229960002920 sorbitol Drugs 0.000 description 12
- 229920001542 oligosaccharide Polymers 0.000 description 9
- 150000003839 salts Chemical class 0.000 description 9
- 239000002253 acid Substances 0.000 description 8
- 229940118179 lovenox Drugs 0.000 description 8
- 239000012071 phase Substances 0.000 description 7
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 7
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 239000000470 constituent Substances 0.000 description 6
- 150000004044 tetrasaccharides Chemical class 0.000 description 6
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical group O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 4
- 239000003055 low molecular weight heparin Substances 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 3
- MSWZFWKMSRAUBD-CBPJZXOFSA-N 2-amino-2-deoxy-D-mannopyranose Chemical compound N[C@@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-CBPJZXOFSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 238000013375 chromatographic separation Methods 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 229960002442 glucosamine Drugs 0.000 description 3
- 150000004676 glycans Chemical class 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 229920001282 polysaccharide Polymers 0.000 description 3
- 239000005017 polysaccharide Substances 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 238000002211 ultraviolet spectrum Methods 0.000 description 3
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 238000006345 epimerization reaction Methods 0.000 description 2
- 150000002302 glucosamines Chemical class 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- WXDKIEQFULDJDL-YNKJQRNTSA-N (2s)-2-amino-3-[(2r,3r,4r)-3,4-dihydroxy-5-[(2s,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]oxypropanoic acid Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](N)C(O)=O)OCC1O[C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 WXDKIEQFULDJDL-YNKJQRNTSA-N 0.000 description 1
- 102000004411 Antithrombin III Human genes 0.000 description 1
- 108090000935 Antithrombin III Proteins 0.000 description 1
- 101100541106 Aspergillus oryzae (strain ATCC 42149 / RIB 40) xlnD gene Proteins 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 238000012369 In process control Methods 0.000 description 1
- AEMOLEFTQBMNLQ-HNFCZKTMSA-N L-idopyranuronic acid Chemical compound OC1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-HNFCZKTMSA-N 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- PRDZVHCOEWJPOB-IVMDWMLBSA-N N-sulfo-D-glucosamine Chemical compound OC[C@H]1OC(O)[C@H](NS(O)(=O)=O)[C@@H](O)[C@@H]1O PRDZVHCOEWJPOB-IVMDWMLBSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 229960005348 antithrombin iii Drugs 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 230000003139 buffering effect Effects 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
- 150000001719 carbohydrate derivatives Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001805 chlorine compounds Chemical class 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960000610 enoxaparin Drugs 0.000 description 1
- 238000010965 in-process control Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- APVPOHHVBBYQAV-UHFFFAOYSA-N n-(4-aminophenyl)sulfonyloctadecanamide Chemical compound CCCCCCCCCCCCCCCCCC(=O)NS(=O)(=O)C1=CC=C(N)C=C1 APVPOHHVBBYQAV-UHFFFAOYSA-N 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical class OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 238000011946 reduction process Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000000020 sulfo group Chemical group O=S(=O)([*])O[H] 0.000 description 1
- 238000000825 ultraviolet detection Methods 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/527—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving lyase
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Saccharide Compounds (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Description
I IN THE MATTER OF an Australian Application corresponding to PCT Application PCT/FR2003/002782 RWS Group Ltd, of Europa House, Marsham Way, Gerrards Cross, Buckinghamshire, England, hereby solemnly and sincerely declares that, to the best of its knowledge and belief, the following document, prepared by one of its translators competent in the art and conversant with the English and French languages, is a true and correct translation of the PCT Application filed under No. PCT/FR2003/002782.
Date: 15 February 2005 C. E. SITCH Deputy Managing Director UK Translation Division For and on behalf of RWS Group Ltd WO 2004/027087 1 PCT/FR2003/002782 Method of determining specific groups forming heparins or low-molecular-weight heparins The subject of the present invention is a method for analysing specific groups constituting heparins or lowmolecular-weight heparins.
During the process for preparing enoxaparin (Lovenox®) (US5,389,618) from pure heparin, the aqueous-phase alkaline depolymerization process produces a partial but characteristic conversion of the glucosamines of the reducing ends of the oligosaccharide chains.
The first step of this conversion consists of a glucosamine-+mannosamine epimerization Toida et al., J. Carbohydrate Chemistry, 15(3), 351-360 (1996)); the second step is a 6-0-desulfation of the glucosamine, leading to the formation of derivatives called "1,6 anhydro" (international patent application W001/29055).
1-Cycization 00
O
Cl This type of derivative is only obtained for oligosaccharide chains whose terminal glucosamine is 6-O-sulfated.
0o The percentage of oligosaccharide chains whose end is modified with a 1,6-anhydro bond is a structural characteristic of the oligosaccharide mixture of Lovenox and it should be possible to measure it.
SThe present invention therefore consists of a method for analyzing heparins, low-molecular-weight heparins and more particularly Lovenox.
r Accordingly, the invention provides a method for analysing heparins or low-molecular-weight heparins to carry out a search for the presence of oligosaccharide chains whose end is modified with a 1,6-anhydro bond, wherein the following steps are carried out: 1 depolymerization of the sample by the action of heparinases, wherein the 1, 6-anhydro residues obtained during the depolymerization reaction are the following: O2Na 2C 0
O
2 O0 OH 0 OH 0
OH
HNS03Na
O
2 Na H 2
C
O
S O NH O 3 Na
OH
O
2 Na 02___ 0 OH 0 OH
OSO
3 Na
NHSO
3 Na 00 O 2 reduction of the depolymerizate c 3 assay by high-performance liquid chromatography, the Schromatographic method used being an anion-exchange chromatography, in 00 which a mobile phase which is transparent in the UV region up to 200 nm is preferably used, and in which a method of detection which makes it possible to selectively detect acetylated sugars is preferably used, said method being carried c out taking as signal the difference between the absorbence at two wavelengths chosen such that the absorptivity of the nonacetylated saccharides cancels out.
SThe method as defined above is therefore characterized in that there is a search for the presence of oligosaccharide chains whose end is modified with a c1,6-anhydro bond ("1,6-anhydro groups").
In particular, the sample to be assayed is first of all exhaustively depolymerized with a mixture of heparinases and in particular heparinase 1 (EC heparnase 2 (heparin lyase II) and heparinase 3 (These enzymes are marketed by the group Grampian Enzymes). International application W088/02400 describes a method of depolymerizing heparin by a heparinase and neutralizing the anticoagulant activity of the heparin contained in a blood sample.
More particularly the method as defined above, is characterized in that the heparinases are in the form of a mixture of heparinase 1 (EC heparinase 2 (heparin lyase II) and heparinase 3 The depolymerizate thus prepared is then treated 4 possible to ascertain that low-molecular-weight heparins do not satisfy the physicochemical characteristics of Lovenox and therefore are different in nature.
The method of assay according to the invention may be applied to the industrial process during in-process control of samples in order to provide standardization of the process for manufacturing Lovenox and to obtain uniform batches.
After enzymatic depolymerization and reduction of the reducing ends, the 1,6-anhydro derivatives of Lovenox exist in 4 essential forms. The subject of the invention is therefore also the method as described above, characterized in that the 1,6-anhydro residues obtained during the depolymerization reaction are the following: HNSOSa OH disaccharide 1 disaccharide 2 OSON8 NHsoNa NHSONa oSoN disaccharide 3 tetrasaccharide 1 All the oligosaccharides or polysaccharides which contain the 1,6-anhydro end on the terminal disaccharide unit and which do not possess a sulfate on the uronic acid of said terminal disaccharide are completely depolymerized by the heparinases and in the form of the disaccharides 1 and 2. On the other hand, when said terminal saccharide contains a 2-O-sulfate on the uronic acid and when it is in the mannosamine form, the 1,6-anhydro derivative is in the form of the tetrasaccharide 1 (form resistant to heparinases).
5 The trisaccharide 1 (see below) is also present in the mixture. It is derived from another degradation process which leads to the structure below (peeling phenomenon observed during the chemical depolymerization of Lovenox).
CO0Na OSOANa 0 OH 0 CO 2 Na\ 0 OH OH OH OSONa
NHSO
8 Na OSO 3 Na trisacchande 1 The other constituents of the mixture are not characteristic solely of Lovenox. There are of course the 8 elementary disaccharides of the heparin chain.
These 8 elementary disaccharides are marketed inter alia by the company Sigma.
Other disaccharides were identified in the mixture by the method according to the invention: the disaccharides AIISgal and AIVSgai which have as origin alkaline 2-0-desulfation of -IdoA(2S)-GlcNS(6S)- and of -IdoA(2S)-GlcNS-, leading to the formation of 2 galacturonic acids. They are not usually present in the original structure of heparin Desai et al., Arch.
Biochem. Biophys., 306 461-468 (1993).
-6- 0 0H aVa AC AIVs N s HNi $0J0a 0lVs
HN
p1 SONa O~oda .O/isa /os~jli HNo
OON
AIIa Ac AMHN sop No o on
NMI
A~fa AC OOS H 'SOPS Ass cop 2"st Ow SThe oioaccharides containing The oligosaccharides containing glucosamines withstand cleavage by heparinases and remain present in the form of tetrasaccharides.
In the case of most low-molecular-weight heparins, the heparin is extracted from pig mucus, and these principal tetrasaccharides are represented below. They are resistant to enzymatic depolymerization and reflect the sequences with affinity for antithrombin III. They are symbolized as follows: AIIa-IIsgiu and AIIa-IVsiu.
YAMADA, K. YOSHIDA, M. SUGIURA, K-H KHOO, H.R. MORRIS, A. DELL, J. Biol. Chem.; 270(7), 4780-4787 (1993) 7 A UA-GlcNAc-GlcA-GlcNS(3,6S) or A A UA-GlcNAc-GlcA-GlcNS(3S) or A Ha-WsJ a The final constituent of the mixture cleaved with heparinases is the glycoserine end AGlcA-Gal-Gal-Xyl- Ser SUGAHARA, H. TSUDA, K. YOSHIDA, S. YAMADA, J. Biol. Chem.; 270(39), 22914-22923 (1995); K. SUGAHARA, S. YAMADA, K. YOSHIDA, P. de WAARD, J.F.G.
VLIEGENTHART; J.Biol.Chem.; 267(3), 1528-1533 (1992).
The latter is generally almost absent from Lovenox (see NMR in Example OOH OH CO,Na OH OH OH OH AGIcA Gal Gal Xyl Ser Another aspect of the invention consists in the chromatography process used for determining the 1,6anhydro groups. First of all, it involves separating the various polysaccharides obtained after depolymerization and treatment with a reducing agent such as NaBH 4 Anion-exchange chromatography (SAX) is the separating method which is most suitable for such a complex mixture.
r 8 Columns filled with a stationary phase of the Spherisorb SAX type having a particle size of 5 pm and a length of 25 cm can be used. All the conventional column diameters between 1 mm and 4.6 mm can be used.
The equipment used may be a chromatograph allowing the formation of an elution gradient with a UV detector, more preferably equipped with an array of diodes in order to be able to produce UV spectra of the constituents and to record complex signals, resulting from the difference between the absorbance at 2 different wavelengths and allowing the specific detection of acetylated oligosaccharides. To allow this type of detection, mobile phases which are transparent in the UV region up to 200 nm are preferable. This excludes conventional mobile phases based on NaCl which have moreover the disadvantage of requiring a passivated chromatogram in order to withstand the corrosive power of the chlorides. The mobile phase used here will be preferably based on sodium perchlorate, but methanesulfonate or phosphate salts may also be used.
The pH recommended for the separation is from 2 to Preferably, a pH in the region of 3 will be used. It is controlled here by adding a salt such as phosphate possessing a buffering power at pH 3 which is better than that of perchlorates.
By way of example, standard chromatographic separation conditions are given below: Solvent A: NaH 2
PO
4 2.5 mM, brought to pH 2.9 by addition of H 3 PO4 Solvent B: NaCO 4 1N- NaH 2
PO
4 2.5 mM, brought to pH 3.0 by addition of H 3 P0 4 The elution gradient may be the following: 9 T 0 min: %B 3; T 40 min: %B 60; T 60 min: %B The subject of the present invention is therefore also a method of analysis as defined above by separation by anion-exchange chromatography, characterized in that the mobile phase which is transparent in the UV region up to 200 nM is used.
The subject of the invention is more particularly a mobile phase as defined above based on sodium perchlorate, methanesulfonate salts or phosphate salts.
Another most important aspect consists in the method of detection.
A method is developed in order to increase the specificity of the UV detection. As nonacetylated polysaccharides all have, at a given pH, a fairly similar UV spectrum, it is possible to selectively detect the acetylated sugars by taking as signal the difference between the absorbance at 2 wavelengths chosen such that the absorptivity of the nonacetylated saccharides cancels out.
In the case below, 202 nm and 230 nm will be chosen as detection and reference wavelengths and the 202-230 nm signal will be noted. The choice of course depends on the pH of the mobile phase (adjustments of a few nm may be necessary so as to be at the optimum of said conditions). The most suitable detector for this technique is the DAD 1100 detector from the company Agilent Technologies. In this case, a double detection will be carried out at 234 nm, on the one hand, and at 202-230 nm, on the other hand. The principle of selective detection of acetylated oligosaccharides is illustrated in the figure below in which the UV spectrum of a sulfated disaccharide Delta Is is compared with that of an acetylated disaccharide Delta la.
10 Std.
600 \AIs o500 400 300 AIa 100
E
0- 200 220 240 240 260 280 The subject of the present invention is therefore also a method of analysis as defined above by separation by anion-exchange chromatography, characterized in that the method of detection makes it possible to selectively detect acetylated sugars..
The subject of the invention is also most particularly a method of analysis as defined above by separation by exchange chromatography, characterized in that the selective detection of acetylated sugars is carried out taking as signal the difference between the absorbance at 2 wavelengths chosen such that the absorptivity of the nonacetylated saccharides cancels out.
The quantification of the 4 1,6-anhydro residues described above requires a sufficient selectivity of the chromatographic system in relation to all the other 11 constituents of the mixture. However, the 2 disaccharides 1 and 2, which are coeluted in general, are poorly resolved with respect to AIIa, especially as the latter is present in the form of its 2 a and anomers.
The identity of the 2 disaccharides 1 and 2 may be easily verified because they form in a few hours at room temperature in an aqueous solution of AIIs brought to pH 13 by addition of NaOH. However, if double detection is used, the acetylated oligosaccharides AIVa, AIIa, VIIIa, Ala, VIIa-IVsgiu and AIIa-IIsg. are easily identifiable.
The causes of splitting of the peaks are the anomeric forms, on the one hand, and to a lesser degree the glucosamine mannosamine epimerization which is partially present for AIIs, AIIIs and AIs when they are in the terminal position in the oligosaccharide chain.
In order to be able to quantify the disaccharides 1 and 2, the sample of low-molecular-weight heparin, depolymerized by heparinases is reduced by the action of NaBH 4
OR
a anomer p anomer This reduction has the advantage of eliminating the a 4- 0 anomerisms by opening of the terminal oligosaccharide ring. The chromatogram obtained is simpler since the anomerisms are eliminated and especially the reduction of AIIa reduces its retention on the column and allows easy assay of the disaccharides 1 and 2.
12 The examples of chromatograms described in Figures 1 and 2 below clearly illustrate these phenomena and the advantages of this method.
Finally, the subject of the invention is also the novel saccharide derivatives obtained using the depolymerization and reduction process, chosen from disaccharide 1, disaccharide 2, disaccharide 3 and trisaccharide 1.
The examples below illustrate the invention without however having a limiting character.
Example 1: The enzymatic depolymerization is carried out for 48 hours at room temperature by mixing 50 pl of a solution containing 20 mg/ml of low-molecular weight heparin to be assayed, 200 pl of a 100 mM acetic acid/NaOH solution at pH 7.0 containing 2 mM calcium acetate and 1 mg/ml of BSA with 50 pl of the stock solution of the 3 heparinases.
The reduction is carried out on 60 pl of the product depolymerized with the heparinases by adding 10 pl of an NaBH 4 solution at 30 g/l in 100 mM sodium acetate prepared immediately before use. It will be noted that the heparinases are stored at -30 0 C. The heparinases are in a buffer solution and their titer is 0.5 IU/ml (composition of the buffer solution: aqueous solution pH 7 of KH 2
PO
4 at a concentration of 0.01 mol/l and supplemented with bovine serum albumin (BSA) at 2 mg/ml).
Example 2: NMR of Disaccharide 3 obtained according to the process described above.
Proton spectrum in D 2 0, 400 MHz, T=298K, 8 in ppm: 3.34 (1H, dd, J=7 and 2Hz, H2), 3.72 (1H, t, J=8Hz, H6), 13 3.90 (1H, m, H3), 4.03 (1H, s, H4), 4.20 (1H, d, J=8Hz, H6), 4.23 (1H, t, J=5Hz, 4.58 (1H, m, 4.78 (1H, m, H5), 5.50 (1H, s, Hi), 5.60 (1H, dd, J=6 and :LHz, 6.03 (1H, d, J=5Hz, H41)].
Example 3 NMR of the Tetrasaccharide 1 obtained according to the process described ab ove.
Proton spectrum in D 2 0, 400 MHz, T=298K, 5 in ppm: 3.15 (1H, s, H2), 3.25 (1H, m, 3.60 (1H, m, between 3.70 and 4.70 (14H, unresolved complex, H3/H4/H6, H2'/H3'/H4'/H5', 4.75 (1H, m, H5), between 5.20 and 5.40 (2H, m, Hi' and 5.45 (1H, m, 5.56 (1H, m, Hi), 5.94 (1H, d, J=5Hz, H4) Example 4: NMR of the Trisaccharide 1 obtained according to the process described above.
Spectrum in D 2 0, 600 MHz, (8 in ppm) 3.28 (1H, in), 3.61 (1H, t, 7Hz), 3.79 (1H, t, 7Hz), 3.95 (1H, d, 6Hz), 4.00 (1H, 4.20 (1H, in), 4.28 (2H, in), 4.32 (1H, d, 4Hz), 4.41 (1H, 4.58 (1H, 4.61 (1H, 4.90 (1H, broad 5.24 (1H, 5.45 (1H, 5.95 (1H, Example NMR of AGlcA-Gal-Gal-Xyl-Ser Spectrum in D 2 0, 500 MHz (8 in ppm): 3.30 (1H, t, 7Hz) 3.34 (1H, t, 8Hz), 3.55 (1H, t, 7Hz), 3.60 (iH, t, 7Hz), between 3.63 and 3.85 (10H, in), 3.91 (2H, in), 3. 96 (1iH, dd, 7 and 2Hz), between 4.02 and 4.10 (3H, in), 4.12 (1H, d, 2Hz), 4.18 (1H, in), 4.40 (1H, d, 6Hz), 4.46 (1H, d, 6Hz), 4.61 (1H, d, 6Hz), 5.29 (1H, d, 3Hz), 5.85 (1H, d, 3Hz).
14 Example 6 Principle of the quantification In the method according to the invention, the widely accepted hypothesis that all the unsaturated oligosaccharides contained in the mixture have the same molar absorptivity, equal to 5500 mol-1.l.cm is made.
It is therefore possible to determine the percentage by weight of all the constituents of the depolymerized mixture in the starting low-molecular-weight heparin.
For the 4 1,6-anhydro derivatives which correspond to the peaks 7,8,13 and 19, the following percentages by weight are obtained: w/w 10 0 443 (Area Area,) [Mw Area, 545-Area,, w/w13 100 Mw.Area CMw, -Area, 1210-Area3 w/w 19 =100. 1210 SMw, -Area Area 7 Areas, Area l3 and Areai 9 correspond to the areas of each of the peaks 7, 8, 13 and 19. The molar masses of each of these 4 compounds are 443, 443, 545 and 1210 respectively. EMw X *Area corresponds to the ratio of the area of each peak of the chromatogram by the molar mass of the corresponding product.
If Mw is the mean mass of the low-molecular-weight heparin studied, the percentage of oligosaccharide chains ending with a 1,6-anhydro ring is obtained in the following manner: The molecular masses of the constituents are the following: 15 Oligosaccharide Oligosaccharide Molecular mass reduction 1 1 741 2 20 401 3 3 734 4 21 461 22 461 6 23 503 7 7 443 8 8 443 9 24 503 25 563 11 26 563 12 27 563 13 13 545 14 28 605 29 1066 16 30 665 17 31 965 18 32 1168 119 119 1210 Nomenclature of the saccharides and correspondence with the peaks according to Figures 1 and 2 IdoA: Gi cA: AGicA: Gal: Xyl: GlcNAc: GlcNS: 2S: 3S: 6S: I1: a-L-Idopyranosyluronic acid; P-D-Glucopyranosyluronic acid; 4,5-unsaturated acid: 4-deoxy-a-Lthreohexenepyranosyluronic acid; D-Galactose; xylose; 2 -deoxy-2 -acetamido-a-D-glucopyranose; 2-deoxy-2 -sulfamido-a-D-glucopyranose; AGlCAI- 3 Gal 0~1-3 GalI3 14 Xyl 16 2: 3: 4: 6: 7: 8: 9: 10: 11: 4 -deoxy-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-acetamido-a-D-glucopyranosyl sodium salt AGlcA3 1 3 Gal 01-3 Galpi..
4 Xyl 1
-O-,CH
2
-COOH
4-deoxy-a-L- threohex-4-enegalactopyranosyl.
uronic acid- -2-deoxy-2-sulfamido- P-D-glucopyranose disodium salt 4 -deoxy-cc-L-threohexenepyranosyluronic acid- 2 -deoxy-2-sulfamido-a-D-glucopyranosyl sodium salt 4 -deoxy-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-acetamido-6--sulfo.a.D-gluco.
pyranosyl disodium salt 4 -deoxy-a-L-threohex-4-enepyranosyluronic acid- E-anhydro-2-deoxy-2-sulfamido- P-D-glucopyranose disodium salt (disaccharide 1) 4 -deoxy-a-L-threohex-4-enepyranosyluronic acid- 6 -anhydro-2-deoxy-2-sulfamido- P-D-mannopyranose disodium salt (disaccharide 2) 4 -deoxy- 2 sulfo--L-threohexenepyranosyl.
uronic acid- 2 -deoxy-2-acetamidoa-D-glucopyranosyl disodium salt 4-deoxy-a-L- threohex-4-enegalactopyranosyluroriic acid- -2-deoxy-2-sulfamido- E-O-sulfo-o-D-glucopyranose trisodium salt 4 -deoxy-cc-L-threohexenepyranosyluronic a-cid- 2 -deoxy-2-sulfamido-6-O-sulfo- P-D-glucopyranosyl trisodium salt 4 -deoxy- 2 -O-sulfo-a-L-threohexenepyranosyl.
uronic acid- -2-deoxy-2-sulfamidoa-D-glucopyranosyl trisodium salt 4-deoxy-2-O-sulfo-a-L- threohex-4-enepyranosyluronic acid- 6-anhydro-2-deoxy- 2 -sulfamido-o.-D-glucopyranose trisodium salt (Disaccharide 3) 12: 13: 17 14 16: 17 7: 18 19: 21: 22: 4 -deoxy- 2 -O-sulfo-a-L-threohexenepyranosyl..
uronic acid- -2-deoxy-2-acetamido- 6-O-sulfo-cL-D-glucopyranosyl trisodium salt 4 -deoxy-ct-L-threohexenepyranosyluronic acid- 2 -deoxy-2-acetamido---sulfox Dguco.
pyranosyl- -1-D-glucopyranosyluronic acid- 2 -deoxy-2-sulfamido-3--sulfo-a-Dglucopyranosyl) pentasodium salt 4 -deoxy- 2 -O-sulfo-a-L-threohexenepyranosy..
uronic acid- -2-deoxy-2-sulfamido- 6-O-sulfo-ct-D-glucopyranosyl tetrasodium salt 4 -deoxy-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-acetamido-6--sulfo-aDglucopyranosyl- -1-D-glucopyranosyluronic acid- -2-deoxy-2-sulfamido-3, 6-di-Q-sulfo-a-Dglucopyranosyl) hexasodium. salt 4 -deoxy-2 sul fo-a-L-threohexenepyranosyluronic acid- -2-deoxy-2-sulfamido- 6 -O-sulf o-D-glucopyranosyl- -2-0-sulf o-a- L-idopyranosyluronic acid hexasodium salt 4 -deoxy- 2 -0-sulfo-a-L-threohexenepyranosyl..
uronic acid- -2-deoxy-2-sulfamido-6-0sulf o-a-D-glucopyranosyl- -2-O-sulf o-x-L'idopyranosyluronic acid- 6-anhydro-2deoxysulfamido-i3-D-mannopyranose, hexasodium salt (tetrasaccharide 1) 4 -deoxy-cx-L-threohexenepyranosyluronic acid- 2 -deoxy-2-acetamido-ca-D-glucitol sodium salt 4 -deoxy-d-L-threohexenepyranosyluronic acid- 2 -deoxy-2-sulfamido-p-D-glucitol disodium salt 4 -deoxy-a-L-threohexenepyranosyluronic acid- (1-44) 2 -deoxy-2-sulfamido-a-D-glucitol disodium salt 4 -deoxy-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-acetamido-6-0-sulfoax-D-glucitol disodium salt 23: 18 24: 26: 27: 28: 29: 4 -deoxy- 2 -0-sulfo-a-L-threohexenepyranosy..
uronic acid- (1-M4 2 -deoxy-2-acetamidoa-D-glucitol disodium salt 4-ex---hehxeeaatprnslrn~ acid- 2 -cleoxy-2-sulf amido-6-...sulf o- !-D-glucitol trisodium salt 4 -deoxy-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-sulfamido-6-0sulfoax-D-glucitol trisodium salt 4 -deoxy- 2 -0-suJlfo-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-sulfamidoa-D-glucitol trisodium salt 4 -deoxy-2 -O-sulfo-a-L-threohexenepyranosy..
uronic acid- 2 -deoxy-2-acetamido- 6-O-sulfo-cx-D-glucitol trisodium salt 4 -deoxy-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-acetamido-6-0osulfoa.Dglucopyranosyl- -1-D-glucopyranosyluronic acid- 2 -deoxy-2-sulfamido-3-0-sulfoa-D-glucitol) pentasodium salt 4 -deoxy- 2 -O-sulfo-a-L-threohexenepyranosyl.
uronic acid- 2 -deoxy-2-sulfamido- E-O-sulfo-a-D-glucitol trisodium salt 4 -deoxy-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-acetamido-6-..sulfo.
a-D-glucopyranosyl- -f-D-glucopyranosyluronic acid- 2 -deoxy-2-sulfamido-3, 6-di- O-sulfo-ac-D-glucitol) hexasodium salt 4 -deoxy- 2 -0-sulfo-a-L-threohexenepyranosyluronic acid- 2 -deoxy-2-sulfamido- E-O-sulf o-a-D-glucopyranosyl. -2-0-sulf oc-L-idopyranosyluronic acid hexasodium salt (form reduced with NaBH 4 31: 32: Figure 1 Chromatographic separation of enoxaparine depolymerized with heparinases before and after reduction with NaBH 4 (signal in fine black: UV at 234 nm; signal in thick black: UV at 202-234 nm) r 19 Figure 2: Chromatographic separation of depolymerized with heparinases before and reduction with NaBH 4 (signal in fine black: 234 nm; signal in thick black: UV at 202-234 nm) heparin after UV at
Claims (9)
1. A method for analysing heparins or low-molecular-weight heparins to carry out a search for the presence of oligosaccharide chains whose end is modified 00oo with a 1,6-anhydro bond, wherein the following steps are carried out: 1 depolymerization of the sample by the action of heparinases, Swherein the 1, 6-anhydro residues obtained during the depolymerization reaction 0C\ are the following: O
2 Na H 2 ,C- 0 SO OH OH OH CO 2 Na HC N 0 O o O Na Na O OH 0 O H OH &O 3 Na OSO 3 Na NHSO3Na OS0Na 2 reduction of the depolymerizate
3- assay by high-performance liquid chromatography, the chromatographic method used being an anion-exchange chromatography, in which a mobile phase which is transparent in the UV region up to 200 nm is which a mobile phase which is transparent in the UV region up to 200 nm is 00 O preferably used, and in which a method of detection which makes it possible to cI selectively detect acetylated sugars is preferably used, said method being carried Sout taking as signal the difference between the absorbence at two wavelengths 00 chosen such that the absorptivity of the nonacetylated saccharides cancels out. 2. The method as defined in claim 1, wherein the heparinases are in the form Sof a mixture of heparinase 1 (EC heparinase 2 (heparin lyase II) and heparinase 3 0 3. The method as defined in claim 1 or 2 wherein the heparin depolymerized C1 by the action of heparinase (depolymerizate) is then subjected to a reducing agent.
4. The method as defined in claim 3, wherein the reducing agent is NaBH 4 or an alkali metal salt of the borohydride anion.
The method as defined in any one of claims 1 to 4, wherein the mobile phase used is based on sodium perchlorate, methanesulfonate salts or phosphate salts.
6. A 1,6-anhydro derivative of formula (disaccharide 1) O 2 Na HC- O 0 OH O OH OH HNSO 3 Na
7. A 1,6-anhydro derivative of formula (disaccharide 2) 2 Na HC-- OH OH NH ONa OH 00
8. A 1 ,6-anhydro derivative of formula (disaccharidle 3) CONa CO 2 Na 0 OH 0 OH OSO0 3 Na NHS03Na
9. A trisaccha ride derivative of formula: 00 0 A method when used for analysing heparins or low-molecular-weight Sheparins to carry out a search for the presence of oligo saccharide chains whose end is modified with a 1,6-anhydro bond wherein the 1, 6-anhydro residues are the following: 0 HO 2 Na 0 2 OH O 0 OH OH HNSO3Na CONa H OHO OH H ONa OH CO 2 Na H W~H OH OSONa a NHSO 3 Na ,OS03Na CO2Na HC OSO 3 Na NHSO 3 Na OSONa substantially as hereinbefore described with reference to Examples 1 and 6, and Figures 1 and 2. AVENTIS PHARMA SA. WATERMARK PATENT TRADE MARK ATTORNEYS P25242AU00
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| FR0211724A FR2844808B1 (en) | 2002-09-23 | 2002-09-23 | METHOD OF DETERMINING SPECIFIC GROUPS CONSISTING OF HEPARINS OR HEPARINS OF LOW MOLECULAR WEIGHT |
| FR02/11724 | 2002-09-23 | ||
| US42248202P | 2002-10-31 | 2002-10-31 | |
| US60/422,482 | 2002-10-31 | ||
| PCT/FR2003/002782 WO2004027087A2 (en) | 2002-09-23 | 2003-09-22 | Method of determining specific groups forming heparins |
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| EP1582531A1 (en) * | 2004-03-24 | 2005-10-05 | Aventis Pharma S.A. | Process for oxidizing unfractionated heparins and detecting presence or absence of glycoserine in heparin and heparin products |
| FR2880951B1 (en) * | 2005-01-19 | 2015-09-18 | Aventis Pharma Sa | METHOD OF ANALYZING OLIGOSACCHARIDES FROM BLOOD PLASMA |
| US8101733B1 (en) | 2006-06-27 | 2012-01-24 | Momenta Pharmaceuticals, Inc. | Methods of evaluating mixtures of polysaccharides |
| BRPI0621470A2 (en) | 2006-12-05 | 2011-12-13 | Glycoscience Lab Inc | therapeutic agent for degenerative arthritis |
| US7790466B1 (en) | 2007-01-26 | 2010-09-07 | Momenta Pharmaceuticals, Inc. | Evaluating mixtures of low molecular weight heparins by chain profiles or chain mapping |
| US7968082B1 (en) | 2007-01-26 | 2011-06-28 | Momenta Pharmaceuticals, Inc. | Evaluating mixtures of low molecular weight heparins by NMR |
| US9139876B1 (en) | 2007-05-03 | 2015-09-22 | Momenta Pharmacueticals, Inc. | Method of analyzing a preparation of a low molecular weight heparin |
| US8435795B2 (en) | 2010-01-19 | 2013-05-07 | Momenta Pharmaceuticals, Inc. | Evaluating heparin preparations |
| WO2012115952A1 (en) | 2011-02-21 | 2012-08-30 | Momenta Pharmaceuticals, Inc. | Evaluating heparin preparations |
| CN103755823B (en) * | 2013-12-12 | 2016-09-28 | 中国海洋大学 | The purification of keratan sulfate and detection method in a kind of chondroitin sulfate |
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