Characterization of currently marketed heparin products: key tests for quality assurance

Abstract

During the 2007-2008 heparin crisis, it was found that the United States Pharmacopeia (USP) testing monograph for unfractionated heparin sodium (UFH) did not detect the presence of the contaminant, oversulfated chondroitin sulfate (OSCS) in heparin. In response to this concern, new tests and specifications were developed by the Food and Drug Administration (FDA) and USP and put in place to not only detect the contaminant OSCS but also to improve assurance of quality and purity of the drug product. Additional tests were also developed to monitor the heparin supply chain for other possible economically motivated additives or impurities. In 2009, a new USP monograph was put in place that includes 500 MHz (1)H NMR, SAX-HPLC, %galactosamine in total hexosamine, and anticoagulation time assays with purified factor IIa or factor Xa. These tests represent orthogonal approaches for UFH identification, measurement of bioactivity, and for detection of process impurities or contaminants in UFH. The FDA has applied these analytical approaches to the study of UFH active pharmaceutical ingredients in the marketplace. Here, we describe results from a comprehensive survey of UFH collected from seven different sources after the 2009 monograph revision and compare these data with results obtained on other heparin samples collected during the 2007-2008 crisis.

Competitive Inhibition of Heparinase by Persulfonated Glycosaminoglycans: A Tool to Detect Heparin Contamination

“…Advanced analytical technologies, including most notably multidimensional NMR, have demonstrated the ability to detect a wide range of potential sulfonated polysaccharides if present within heparin, 6 since initial efforts to detect OSCS in heparin employ a variety of strategies, including HPLC, 9,10 bioassays, 11 and spectroscopy. 12,13 However, development of additional, orthogonal assays that can rapidly and sensitively detect the presence of potential contaminants, if present, is warranted.…”

Linear polyalkylamines as fingerprinting agents in capillary electrophoresis of low‐molecular‐weight heparins and glycosaminoglycans

“…A number of methods have been reported in the literature for this purpose including 1 H NMR 18, 29, 42–44 and SAX‐HPLC 12, 29, 42. In fact, the FDA in conjunction with the US Pharmacopea (USP) has added both SAX‐HPLC and 1 H NMR to its protocol for UFH quality 45, 46 (http://www.usp.org/pdf/EN/hottopics/heparinSodiumMonograph.pdf). CE, PAGE, and two‐dimensional NMR techniques have also been proposed for the analysis of UFH with respect to contamination 15, 18, 44.…”

Characterization of currently marketed heparin products: Adverse event relevant bioassays

“…Panel B shows the effects of heparin API (1000 g/mL) from 6 manufacturers (API 1-19, 1-3 lots each), LMWH from 3 sources (L1-4 dalteparin, L5-7 tinzaparin, L8-10 enoxaparin), USP heparin sodium identity reference standard and OSCS on KK activity. Panel C shows effects of authentically contaminated heparin (1000 g/mL) (C5-C10, % OSCS determined by SAX-HPLC [25]), USP heparin and OSCS on KK activity. KK activity was determined following incubation of plasma 37 • C for 50 min with GAGs or FXIIa followed by addition of Chromogenix (1 mM) for an additional 10 min then measuring the absorbance at 405 nm.…”

“…During 2008, the agency tested a number of authentically contaminated heparin sodium APIs (for review [25]). Five samples were selected with OSCS contamination levels from 1.3 to 24.3% (vida SAX-HPLC) were tested in triplicate and found to have significantly elevated maximal UV absorption from protamine-GAG complexes (OD 280 0.38-0.74) compared to heparin sodium (0.27).…”

“…Panel B shows a measure of complex formation of authentically contaminated heparins (C5-C10, % OSCS determined by SAX-HPLC[25]), USP heparin and OSCS with protamine. Protamine complexes were determined following incubation of 0.15 mg/mL protamine with 1 mg/mL heparin APIs or OSCS for 2 h at room temp and measuring absorbance at 280 nm.…”