Rapid O-glycan release from glycoproteins using a monolith-based glycosidase microreactor

• A chemically/mechanically stable monolith was polymerized in a 50 µm I.D. capillary. • Glycans were rapidly released from desialylated fetuin within a 3 s residence time. • The IMER releases O -glycans as efficiently as the in-solution digestion. Fast, efficient and simple selective O -glycan release methods are urgently needed for accurately analyzing O -glycans of therapeutic proteins or glycosylation-based biomarkers. In this study, a polymeric monolith-based microreactor with immobilized O -glycosidase inside a capillary has been developed. The photopolymerization of a poly (glycidyl methacrylate- co -poly (ethylene glycol) diacrylate) monolith in a capillary with tens of nanoliters volume (50 μm I.D. × 1 cm) was optimized. It exhibited a relatively high permeability (2.29 × 10 –13 m 2 ), and good mechanical and chemical stabilities. Two different immobilization chemistries to attach O -glycosidase and several digestion protocols were tested and compared. We showed that Shiff base and Epoxy chemistries for O -glycosidase immobilization led to similar digestion efficiencies regarding the quantity of released O -glycans. The performance of the immobilized enzyme reactor (IMER) was assessed on asialofetuin used as a substrate for the release of O -glycans. In-IMER digestion was able to release 56 % of O -glycans in <3 s, while the enzyme in-solution digestion required at least one hour to achieve the same release efficiency. The capillary format monolith proves the route to fulfil on-line coupling between O -glycan release and glycan separation analysis.