DeBono, N. J.; Cain, J. A.; Lin, C.-H.; Packer, N. H.; Packer, N.; Moh, E. S. X.

Controlling protein glycosylation as a critical quality attribute of biopharmaceuticals remains challenging when glycosylation is coupled to cellular production systems. Here, we present a proof-of-concept glycosyltransferase immobilised enzyme reactor (IMER) housed within a 3D-printed column that enables directed post-production glycan modification of purified glycoproteins. Using {beta}-1,4-galactosyltransferase ({beta}4GalT1-IMER) and -2,6-sialyltransferase (ST6Gal1-IMER) immobilised on Ni-NTA resin, the IMER achieved near-complete galactosylation and substantial sialylation of partially deglycosylated bovine fetuin N-glycans with their respective substrates with a maximum substrate-enzyme contact time of four minutes. Isomeric-level analysis revealed arm-specific addition preferences for both enzymes, consistent with known specificities. The modular IMER design permits sequential connection of individual enzyme chambers, potentially offering a scalable, plug-and-play platform for constructing defined glycan structures on recombinant glycoprotein therapeutics.