USP compendial methods for analysis of heparin: chromatographic determination of molecular weight distributions for heparin sodium

Heparin is a polysaccharide product isolated from glycosaminoglycans of porcine mucosa (or occasionally other tissues and species). It is a linear non-uniform polymer consisting of alternating glucosamine and uronic acid monosaccharide residues and is highly sulfated. Heparin sodium drug product (HP) used in medicine consists of chains with molecular weight (MW) ranging from under 5,000 to over 50,000. Although HP has been used as an injectable antithrombotic medicine for more than 70 years, many aspects of its structure and purity, including its MW, have not been specified by public standards until recent years. In 2008, a number of HP lots associated with severe adverse effects, including fatalities, were found to have been contaminated with oversulfated chondroitin sulfate. This incident led to thorough revision of compendial standards worldwide. In the USA, the Food and Drug Administration (FDA) encouraged the inclusion of enhanced standards for purity and identity in the relevant monographs of the United States Pharmacopeia (USP) including acceptance criteria for MW distribution

Molecular Weights of Bovine and Porcine Heparin Samples: Comparison of Chromatographic Methods and Results of a Collaborative Survey

In a collaborative study involving six laboratories in the USA, Europe, and India the molecular weight distributions of a panel of heparin sodium samples were determined, in order to compare heparin sodium of bovine intestinal origin with that of bovine lung and porcine intestinal origin. Porcine samples met the current criteria as laid out in the USP Heparin Sodium monograph. Bovine lung heparin samples had consistently lower average molecular weights. Bovine intestinal heparin was variable in molecular weight; some samples fell below the USP limits, some fell within these limits and others fell above the upper limits. These data will inform the establishment of pharmacopeial acceptance criteria for heparin sodium derived from bovine intestinal mucosa. The method for MW determination as described in the USP monograph uses a single, broad standard calibrant to characterize the chromatographic profile of heparin sodium on high-resolution silica-based GPC columns. These columns may be short-lived in some laboratories. Using the panel of samples described above, methods based on the use of robust polymer-based columns have been developed. In addition to the use of the USP’s broad standard calibrant for heparin sodium with these columns, a set of conditions have been devised that allow light-scattering detected molecular weight characterization of heparin sodium, giving results that agree well with the monograph method. These findings may facilitate the validation of variant chromatographic methods with some practical advantages over the USP monograph method

Modernization of Enoxaparin Molecular Weight Determination Using Homogeneous Standards

Enoxaparin is a low-molecular weight heparin used to treat thrombotic disorders. Following the fatal contamination of the heparin supply chain in 2007–2008, the U.S. Pharmacopeia (USP) and U.S. Food and Drug Administration (FDA) have worked extensively to modernize the unfractionated heparin and enoxaparin monographs. As a result, the determination of molecular weight (MW) has been added to the monograph as a measure to strengthen the quality testing and to increase the protection of the global supply of this life-saving drug. The current USP calibrant materials used for enoxaparin MW determination are composed of a mixture of oligosaccharides; however, they are difficult to reproduce as the calibrants have ill-defined structures due to the heterogeneity of the heparin parent material. To address this issue, we describe a promising approach consisting of a predictive computational model built from a library of chemoenzymatically synthesized heparin oligosaccharides for enoxaparin MW determination. Here, we demonstrate that this test can be performed with greater efficiency by coupling synthetic oligosaccharides with the power of computational modeling. Our approach is expected to improve the MW measurement for enoxaparin