Fractionation of Oligosaccharide Nucleoside Mixtures by Single Pass Nano-Diafiltration

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 The application of a novel 3D-printed double-membrane nanofiltration module for the purification of saccharides from enzymatic reaction mixtures was reported in this study.In a first step, a suitable nanofiltration membrane was characterized.Low recovery rates after single-pass nano-diafiltration in the 3D-printed module were overcome by the application of a static mixer, which highly increased product recovery. The notably low purities at high diavolume ratios were traced back to the buffer system, and with an investigation of the influence of different buffer systems on the separation process, we were able to achieve high product purities.The results are an important contribution to designing novel purification processes for mixtures resulting from continuous enzymatic synthesis.