Separation of hyaluronan oligosaccharides by the use of anion-exchange HPLC

Abstract

There has been a recent surge of interest in hyaluronan (I-IA), a glycosaminoglycan. Hyaluronan is a linear polymer composed of the repeating disaccharide unit [(l + 3)-0-(2-acetamido-2-deoxy-&D-gIucopyranosyl)-(1 + 4&0-P-D-glucopyranuronosyl]. Originally thought to serve only a structural function in connective tissue, HA has now been found to play important roles in such diverse functions as cell-cell recognition’, limb, lung, and brain morphogenesis’,’ and as a potential regulator of tumor angiogenesis 3. It has also found biomedical applications in ophthalmic surgery and wound healing4. In the course of our Nh4R investigations of the solution conformation of hyaluronan as a function of chain length, we have developed a rapid, simple method for the preparation and analysis of monodisperse hyaluronan oligosaccharides. With this method we are able to separate HA oligomers of between 2 and 20 disaccharide units at a moderate pH so that chemical modification of the HA is unnecessary. Several methods for the separation of hyaluronan oligosaccharides already exist. Preparation of HA fragments has traditionally been accomplished by gel-permeation chromatography coupled with uranic acid analysis for peak identification5,6. Although this method is capable of separating HA oligomers up to 23 disaccharide units, it is tedious and time consuming, especially when used to prepare relatively large (> 10 mg) quantities of each fragment. HPLC methods including normalphase7, reversed-phase ion-pair’, and weak-anion exchange’ have also been used for the analysis and separation of HA oligosaccharides of varying length. Similar methods10-16 have been used to separate HA oligosaccharides from other glycosaminoglycans. However, the largest fragment that any of the published HPLC methods has been able to separate is six disaccharide units’. The development of a gel electrophoresis method allowed the analysis of oligosaccharides between 7 and 250 repeating disaccharide units 17*18 A capillary zone electrophoresis method has .