**5. Glycoengineering: finding the sweet spot with complex sugars**

Glycosylation is a post-translational protein modification which involves the incorporation of complex sugar molecules (N-linked or O-linked glycans) to specific amino acid residues. It is a common but complex process due to the potential heterogeneity of glycan composition and site occupancy [52]. Antibody glycosylation is

dependent on the expression system, as post-translation modifications differ between cell types and production platforms [41]. Glycoengineering approaches in plants for immune modulation usually focuses on changes in galactosylation, fucosylation and sialylation [53]. The glycans associated with an antibody can significantly impact functional characteristics and is a parameter which must be closely monitored in potential therapeutic antibodies [54].

The singular N-linked Fc glycosylation site in IgG has been extensively studied and engineered for a tailored immune response. For example, removing the core fucose increases antibody-dependent cell-mediated cytotoxicity (ADCC) by facilitating the antibody's binding to FcγRIIIA [55]. Conversely, the inflammatory response mediated by IgG effector functions can be reduced by using sialylated glycans, as this impairs complement-dependent cytotoxicity [55]. It is clear even a single glycan site is influential to IgG function. In comparison, a fully assembled SIgA complex can harbour as many as 26 sites.

Every component of SIgA has potential N-linked glycosylation sites: 4 to 9 per IgA monomer, 1 on the joining chain and 7 on the secretory component. In addition, the IgA1 subtype has an extended hinge region with up to 6 clustered O-glycan sites which make investigations into glycosylation status and the resulting effect on function difficult [56]. Despite this, it is clear that glycosylation is important for effective SIgA function. For instance, SIgA with unglycosylated secretory component is subject to rapid breakdown in the gut [57]. Indeed, the glycan repertoire of IgA is markedly different to that of the well-established IgG and requires extensive further study for levels of familiarity to be comparable [58].
