Efficient methods for isolating N-glycans are essential to understanding the functions and characteristics of the entire N-glycome. Enzymatic release using PNGaseF is the most effective approach for releasing mammalian N-glycans for analytical purposes. However, the use of PNGaseF for preparative N-glycan isolation is precluded due to the enzyme′s cost and limited stability. In this work, we develop a PNGaseF heterogeneous biocatalyst for the preparative isolation of N-glycans from natural sources. By controlling the immobilization conditions, 100–51 % of offered PNGaseF is immobilized on aldehyde-functionalized agarose porous microbeads through distinct protein orientations, achieving different performances. The enzyme orientation through the N-terminus provides the best activity/operational stability balance, being 20 % more efficient than that randomly oriented. This active and stable heterogeneous biocatalyst eases its application in a packed bed reactor (PBR) for continuous release of free N-glycans from a model glycoprotein. This PBR processes 1 g of ovalbumin from chicken egg white to isolate 95 % of its N-glycans upon operating the PBR for 7 days. Finally, by tuning the flow rate, we can control the profile of N-glycans isolated due to different enzyme kinetics for the deglycosylation reactions. In-line methodologies to isolate N-glycans open new paths for more sustainable protocols to prepare relevant glycans. 

DOI: 10.1002/cssc.202402346