Oversulfated chondroitin sulfate is a contaminant in heparin associated with adverse clinical events

Abstract

Heparin has been used clinically as an anticoagulant for over 60 years. Typically isolated from porcine intestine, heparin is a mixture of dimeric glycosidic sequences generating complex polysaccharide glycosaminoglycan chains. Recently, certain lots of heparin have been associated with an acute, rapid onset of significant side effects indicative of an allergic-type reaction. To identify potential causes for this serious rise in side effects, we examined lots of heparin that correlated with adverse events using orthogonal high resolution analytical techniques. Through comparison of these results with those obtained on reference lots, suspect lots were found to contain a highly sulfated chondroitin sulfate contaminant. Through detailed structural analysis, the contaminant was found to contain a disaccharide repeat unit of glucuronic acid linked β1→3 to a β-galactosamine. Surprisingly, the disaccharide unit contains an unusual sulfation pattern and is sulfated at the 2-O and 3-O positions of the glucuronic acid as well as at the 4-O and 6-O positions of the galactosamine. The presence of such a contaminant could elicit a biological response as highly sulfated polysaccharides, such as dextran sulfate, are known to be potent mediators of the immune system. Given the nature of the contaminant, traditional screening tests -such as those present as part of the current United States Pharmacopeia heparin monograph -cannot differentiate between affected and unaffected lots. Our analysis suggests effective screening methods that can be employed to determine whether or not heparin lots contain the contaminants reported here.

Contaminated Heparin Associated with Adverse Clinical Events and Activation of the Contact System

“…4 The identity of the contaminant was confirmed by means of multiple orthogonal techniques, including multidimensional nuclear magnetic resonance (NMR), enzymatic digestion followed by high-performance liquid chromatography, and liquid chromatography-mass spectrometry. 4 After identification of the contaminant as OSCS, a synthetic standard was generated through chemical sulfonation of chondroitin sulfate A and was exhaustively characterized to ensure authenticity, as previously described. 4 The synthetic OSCS was used in spiking experiments to qualify the analytic procedures (especially one-dimensional proton NMR, described below) to determine limits of detection and to establish accurate quantification.…”

“…OSCS was purified to homogeneity from a lot of heparin that was known to be contaminated, as previously described. 4 Briefly, OSCS-contaminated heparin was subjected to anion-exchange chromatography followed by alcohol precipitation to isolate the contaminant. 4 The identity of the contaminant was confirmed by means of multiple orthogonal techniques, including multidimensional nuclear magnetic resonance (NMR), enzymatic digestion followed by high-performance liquid chromatography, and liquid chromatography-mass spectrometry.…”

“…4 Briefly, OSCS-contaminated heparin was subjected to anion-exchange chromatography followed by alcohol precipitation to isolate the contaminant. 4 The identity of the contaminant was confirmed by means of multiple orthogonal techniques, including multidimensional nuclear magnetic resonance (NMR), enzymatic digestion followed by high-performance liquid chromatography, and liquid chromatography-mass spectrometry. 4 After identification of the contaminant as OSCS, a synthetic standard was generated through chemical sulfonation of chondroitin sulfate A and was exhaustively characterized to ensure authenticity, as previously described.…”

“…4 In addition, dermatan sulfate, a known impurity of heparin, was found in selected samples. Both heparin and chondroitin sulfate are members of the glycosaminoglycan family of complex polysaccharides; heparin contains a disaccharide repeat unit of glucuronic-iduronic acid linked to glucosamine, and chondroitin sulfate contains a disaccharide repeat unit of glucuronic acid linked to galactosamine.…”

Structure versus anticoagulant and antithrombotic actions of marine sulfated polysaccharides

“…It is also well known to present several limitations due to collateral effects and limited sources of material (Mourão, 2004). The situation has been complicated recently due to the alarming discovery of heparin preparations that have been contaminated with oversulfated chondroitin sulfate (Guerrini et al, 2008). This contaminant induces hypotension associated with kallikrein release when administered by intravenous injection (Kishimoto et al, 2008).…”