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Quantitative PCR and Disaccharide Profiling to Characterize the Origin of Low Molecular Weight Heparins
Author(s) -
Houiste Celine,
Auguste Cecile,
Anger Pascal,
Brin JeanFrancois
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.6.lb89-d
Subject(s) - depolymerization , chemistry , disaccharide , heparin , low molecular weight heparin , chromatography , biochemistry , organic chemistry
Low molecular weight heparins (LMWHs) are now widely used in the management of venous and arterial thrombosis. These drugs are obtained by controlled enzymatic and chemical depolymerization of porcine mucosal heparin (PMH). To ensure the origin of the raw material used for the manufacture of LMWHs, quantitative PCR (Q‐PCR) is used for acceptance of crude PMH. Heparinase depolymerized disaccharide profile and stable isotope patterns are also used to confirm the porcine origin of these products. Enoxaparin, the most widely used LMWH, contains distinct structural and molecular oligosaccharide profiles that, with the origin of the starting material, add to its uniqueness. Although several generic versions of enoxaparin have been proposed, specific release criteria including the origin of the crude heparin utilized for their production have not been established. A Q‐PCR method has been developed to ensure the porcine origin of the crude heparin with a DNA detection limit of 7 pg/mg for bovine and 70 pg/mg for ovine contaminants. This method is routinely used as an acceptance criterion in the manufacture of enoxaparin for the non US batches so far. In addition, disaccharide profiling after exhaustive depolymerization with heparinase to determine the dIIIs to dIa ratio is used to discriminate porcine from bovine material. Additional approaches such as principal component analysis and stable isotope ( 13 C, 15 N, 18 O, 35 S) can be used to discriminate porcine from ovine heparin. Consistent with the availability of newer technology, these methods should be required as acceptance criteria for ensuring porcine origin of the starting material used in the manufacture of branded and generic LMWHs.