Fractionation of Oligosaccharide Nucleoside Mixtures by Single Pass Nano-Diafiltration

Abstract

Glycans, a diverse group of complex oligosaccharides, are critical to human physiology and hold significant potential in medical applications and as food additives. However, their synthesis by glycosyltransferases produces intricate mixtures comprising saccharides, nucleosides, and reaction buffer components, posing substantial challenges for downstream processing and purification. This study aims to establish a continuous, single-pass nanofiltration process for purifying oligosaccharide-nucleoside mixtures using a novel dual-membrane module. We investigated the influence of a static mixer, along with varying flow rates for both the diafiltration and feed streams, on the recovery rate and purity of the final product. Measurements using ESI-MS assessed product recovery and purity, while buffer removal was monitored through online conductivity measurement. The results demonstrate that incorporating a static mixer nearly doubled the saccharide recovery rate, achieving product purities exceeding 99% and 95%, along with high product recovery rates. Additionally, the reaction buffer system was found to significantly impact the overall process performance. These findings suggest that our novel dual-membrane module can be effectively utilized for the purification of enzymatically synthesized glycan products.

Summary