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

A two-dimensional chromatography method for monitoring the formation of oli gosaccharides produced by heparinase-catalyzed depolymerization of heparin is reported. In the first step of the two-dimensional method, the depolymer ized heparin is size-fractionated by high-performance get permeation chroma tography (HPGPC). The size-uniform fractions are then separated on the basi s of charge by strong anion-exchange (SAX) HPLC on a high resolution CarboP ac PA1 column. To demonstrate application of the two-dimensional product-pr ofile analysis method, data are presented for the heparinase I-catalyzed de polymerization of heparin in the absence and presence of histamine, a ligan d 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 PAI columns for the semipreparative scale separation o f oligosaccharides in size-uniform fractions isolated from depolymerized he parin by low-pressure (gravity flow) GPC is also reported. The semi-prepara tive scale CarboPac PAI column gives high resolution and excellent reproduc ibility after repeated use over an extended period of time, making it possi ble to reliably combine fractions from multiple separations. The oligosacch arides are eluted from the CarboPac PA I column with a NaCl gradient at rel atively low pH (3 or 7) where they are stable. An efficient two-step proced ure is described for desalting oligosaccharides separated by SAX-HPLC. (C) 2001 Elsevier Science B.V. All rights reserved.