Isolation and Characterization of Contaminants in Recalled Unfractionated Heparin and Low-Molecular-Weight Heparin

Abstract

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.

A New Approach for Heparin Standardization: Combination of Scanning UV Spectroscopy, Nuclear Magnetic Resonance and Principal Component Analysis

“…Subsequently, several other groups published papers on the same subject using a wide variety of methods [23], [24], [25], [26], [31]. More recently, the contaminant was claimed to compromise not only OSCS but a mixture of other chemically sulfated GAGs, probably arising from waste generated throughout the heparin production process [22].…”

“…Semi-synthetic (SOSCS) and isolated heparin contaminant (IHC) were prepared as previously described [25]. Pure heparin samples were a gift from Dr. Valentina Baigorria (Kim Master Produtos Químicos Ltd, Brazil) and Gentium SpA (Villa Guardia (CO), Italy).…”

“…Once again, using NMR and other analytical methods, the major contaminant present in heparin batches related to adverse clinical events was identified as an oversulfated chondroitin sulfate (OSCS) [21]. Several papers were then published regarding analytical approaches to assess heparin purity [13], [22], [23], [24], [25], [26]. However, many of these methods are expensive, time consuming and/or require access to sophisticated and costly instrumentation and facilities, making them difficult to use for producers and regulatory authorities in many locations.…”

Pharmacology of Heparin and Related Drugs

“…The addition of EDTA corrects the chemical shift and linewidth variations induced by divalent cations (McEwen, 2010), and the calcium effects can be utilized to aid the identification and quantification of OSCS (McEwen et al, 2009 (Hashii et al, 2010) can be used. An extra dimension of specificity can be added to chromatographic separations of heparin and OSCS by means of nitrous acid digestion that depolymerizes heparin while leaving NA chondroitins untouched (Viskov et al, 2009a).…”

Analysis and characterization of heparin impurities

“…The complete 1 H NMR assignment of OSCS has been reported in several recent publications [1, 31, 52, 53] and in the original work by Maruyama et al [27]. It is also important to note that Holzgrabe et al published 1 H NMR spectra of heparin in 1998 which already contained the resonances of OSCS [54].…”