Abstract
Heparin is a widely-used intravenous anticoagulant comprising a complex mixture of highly-sulfated linear polysaccharides of repeating sequences of uronic acids (either iduronic or glucuronic) 1->4 linked to D-glucosamine with specific sulfation patterns. Preparation of crude heparin from mammalian mucosa involves protease digestion with alcalase under basic conditions (pH ≥ 9) and high temperature (>50°C) and also oxidation. Under such conditions, side reactions including the ubiquitous 2-O desulfation occur on the heparin backbone yielding non-endogenous disaccharides within polysaccharide chains. Whatever the process used for its manufacture, some level of corresponding degradation impurities is therefore expected to be found in heparin and the derived Low Molecular Weight Heparins. These impurities should be monitored to control the quality of the final therapeutic product. Two anion exchange chromatography techniques were used to analyze heparin samples exhaustively or partially depolymerized with heparinases and determine the proportions of non-endogenous disaccharides generated by side reactions during the manufacturing process (epoxides and galacturonic moieties). We also present data from a case study of marketed heparin. Current heparin sodium monographs do not directly address process impurities related to modification of the structure of heparin. Although desulfation reduces the overall biological potency, we found that heparin with an average of one modified disaccharide per chain can still comply with the USP or Ph. Eur. heparin sodium monographs requirements. We have implemented disaccharide analysis to monitor the quality of this product on a risk basis.
Highlights
Heparin is a key pharmaceutical product in the thrombosis field
We further present the findings of an investigation of these structural modifications undertaken as part of the follow-up on an out of specification (OOS) heparin batch
Calibration was performed using a broad standard table associated with the internallyqualified standard, which is similar to the procedure described in the current United States Pharmacopeia (USP) Heparin Sodium monograph [1]
Summary
Heparin is a key pharmaceutical product in the thrombosis field. It can be used therapeutically as unfractionated heparin (UFH), or as the starting material in the preparation of low-molecularweight heparins (LMWHs). Different process structural impurities [4,5,6,7] impacting or not anti-IIa are known and either systematically present or present at sometimes significant levels, we here only focus on those resulting from the ubiquitous 2O-desulfation. We review this key side reaction and discuss how disaccharide and oligosaccharide analyses using strong anion-exchange (SAX) chromatography after heparinase digestions can be used to monitor these. The discussed techniques can be used to further improve quality control at the raw/crude/pure stages of heparin production
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