Asymmetric <i>N</i>‑Glycosylation\nin the Tailpiece of Recombinant IgA1

Here, we employed\na variety of mass spectrometry (MS)-based approaches,\nboth (glyco)peptide-centric and protein-centric, to resolve the complex\nglycoproteoform landscape of recombinant IgA1 produced in HEK293 cells.\nThese key immunoglobulins harbor several <i>N</i>- and <i>O</i>-glycosylation sites, making them considerably more heterogeneous\nthan their IgG counterparts. We provide quantitative data on the occupancy\nand glycan composition for each IgA1 glycosylation site. Combining\nall data, we revealed that IgA1 molecules consist of at least three\ndistinct populations with varying <i>N</i>-glycosylation\nsite occupancies at the C-terminal tailpiece, namely, one with both\nglycosylation sites occupied, another with both glycosylation sites\nunoccupied, and a third asymmetric population with one glycosylation\nsite occupied and the other unoccupied, challenging the prevailing\nacceptance that IgA1 <i>N</i>-glycosylation is symmetrical.\nThis finding is significant, given that the tailpiece is involved\nin interactions with the J-chain and the Polymeric Immunoglobulin\nReceptor, and in general as antibody glycosylation is a quality attribute\nthat needs to be carefully monitored, as the presence and nature of\nthese modifications can affect the antibody’s efficacy, lifetime,\nstability, and binding and/or neutralizing capacities. Optimizing\nstrategies to produce recombinant IgA1 requires efficient and specific\nquality control analytical strategies, as presented here, which is\nessential for therapeutic IgA1-based antibody development. We expect\nthat the integrated MS-based strategy presented here may be beneficial\nto comprehensively characterize the glycoproteoform profiles of IgA1-based\ntherapeutics, thereby improving their production and optimization\nprocesses and facilitating the pathway to bring more IgA1-based therapeutics\ninto clinical applications.