Isolation and characterization of contaminants in recalled unfractionated heparin and low-molecular-weight heparin.
Recently, a contaminant was found in some clinically used unfractionated heparin (UFH) preparations. Administration of this UFH was associated with an increased risk of developing a wide range of adverse effects including death. To further investigate the chemical profile of the contaminant, contaminated batches of UFH were treated by exhaustive nitrous acid depolymerization followed by methanol precipitation to remove heparin oligosaccharides. Because contaminated heparins may have been used as starting material in the production of low-molecular-weight heparins (LMWHs), a similar procedure was carried out using an experimental batch of enoxaparin prepared from contaminated heparin. While high-pressure liquid chromatography (HPLC) analysis of contaminated heparin did not distinguish the presence of the contaminant, it could readily be observed as a high-molecular weight shoulder in the elution profile of contaminated enoxaparin. Digesting contaminated heparin with heparinase-I prior to HPLC analysis showed the presence of a nondigestible component (15%-30% of the mixture). This contaminant was also resistant to degradation by chondroitinases A, B, and C. Proton nuclear magnetic resonance (NMR) indicated that the contaminant was oversulfated chondroitin sulfate (OSCS). Size-exclusion chromatography indicated that the mean molecular weight of the OSCS was 16.8 kD, comparable to that of a synthetic porcine cartilage OSCS preparation that was used as a reference material (17.2 kD). While varying degrees of high-molecular weight dermatan sulfate and other minor impurities were detected, OSCS appeared to be the major contaminant in these preparations. The process involved in the production of enoxaparin does not significantly degrade OSCS.
Viskov
Christian
C
sanofi-aventis, 13 Quai Jules Guesde, Vitry sur Seine, France. Christian.Viskov@sanofi-aventis.com
Bouley
E
E
Hubert
P
P
Martinez
Celine
C
Herman
F
F
Jeske
Walter
W
Hoppensteadt
Debra
D
Walenga
Jeanine M
JM
Fareed
Jawed
J
D000818:Animals; D000925:Anticoagulants / Q000032:analysis*; D019765:Chondroitin ABC Lyase; D002809:Chondroitin Sulfates / Q000737:chemistry / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D004340:Drug Contamination; D017984:Enoxaparin / Q000032:analysis; D019763:Heparin Lyase; D006495:Heparin, Low-Molecular-Weight / Q000032:analysis*; D000432:Methanol; D008970:Molecular Weight; D009608:Nitrous Acid; D019906:Nuclear Magnetic Resonance, Biomolecular; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D012754:Sharks; D013552:Swine
D000925:Anticoagulants; D017984:Enoxaparin; D006495:Heparin, Low-Molecular-Weight; D009844:Oligosaccharides; D002809:Chondroitin Sulfates; D019765:Chondroitin ABC Lyase; D019763:Heparin Lyase; D009608:Nitrous Acid; D000432:Methanol