Quantitative compositional analysis of heparin using exhaustive heparinase digestion and strong anion exchange chromatography

Heparin is a linear sulfated polysaccharide widely used therapeutically as an anticoagulant. It is also the starting material for manufacturing low-molecular-weight heparins (LMWH). Quality control of heparin and LMWH is critical to ensure the safety and therapeutic activity of the final product. However due to their complex and heterogeneous structure, orthogonal analytical techniques are needed to characterize the building blocks of heparin. One of the state-of-the-art methods for heparin analysis is based on complete enzymatic digestion using a mixture of heparinases I, II, and III, followed by the separation of the resulting oligosaccharides by liquid chromatography. The European Pharmacopoeia strong anion-exchange chromatographic method, used to quantify 1,6-anhydro derivatives in enoxaparin, is here applied to the analysis of the heparin building blocks. Their quantification, namely the determination of their average w/w percentage in the heparin chain, is obtained after identification of all components including glycoserine derivatives and 3-O sulfated di- and tetrasaccharides. This work therefore provides a comprehensive overview of the building blocks of unfractionated heparin, including those chemically modified by the manufacturing process, either within the polysaccharide chain or at its reducing end.