Chromatographic methods for product-profile analysis and isolation of oligosaccharides produced by heparinase-catalyzed depolymerization of heparin

Abstract

A two-dimensional chromatography method for monitoring the formation of oligosaccharides produced by heparinase-catalyzed depolymerization of heparin is reported. In the first step of the two-dimensional method, the depolymerized heparin is size-fractionated by high-performance gel permeation chromatography (HPGPC). The size-uniform fractions are then separated on the basis of charge by strong anion-exchange (SAX) HPLC on a high resolution CarboPac PA1 column. To demonstrate application of the two-dimensional product-profile analysis method, data are presented for the heparinase I-catalyzed depolymerization of heparin in the absence and presence of histamine, a ligand that binds site-specifically to heparin. Results from the two-dimensional analysis indicate that histamine alters the extent of depolymerization and the product-profiles for the tetrasaccharide and hexasaccharide fractions. The use of CarboPac PA1 columns for the semi-preparative scale separation of oligosaccharides in size-uniform fractions isolated from depolymerized heparin by low-pressure (gravity flow) GPC is also reported. The semi-preparative scale CarboPac PA1 column gives high resolution and excellent reproducibility after repeated use over an extended period of time, making it possible to reliably combine fractions from multiple separations. The oligosaccharides are eluted from the CarboPac PA1 column with a NaCl gradient at relatively low pH (3 or 7) where they are stable. An efficient two-step procedure is described for desalting oligosaccharides separated by SAX-HPLC.