Characterization of <i>O</i>-Glycosylation and <i>N</i>-Glycosylation in Bispecific Antibodies and Its Importance in Therapeutic Antibody Development

<b>Background/Objectives</b>: This study comprehensively characterized the <i>O</i>- and <i>N</i>-glycosylation profiles of bispecific antibodies (BsAbs) via advanced analytical techniques to evaluate their structural and functional implications. <b>Methods</b>: High-resolution MS revealed <i>O</i>-xylosylation at Ser468 within the (G4S)4 linker peptide, which was identified as xylose with a molecular weight of 132.042 Da. HILIC-HPLC analysis of <i>N</i>-glycosylation revealed glycan species engineered to eliminate Fc effector functions. <i>O</i>-glycosylation analysis via β-elimination followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) identified xylose as the predominant glycan. <b>Results</b>: <i>O</i>-xylosylation does not affect the binding of BsAbs to either antigen Programmed Death-1 (PD-1) or Vascular Endothelial Growth Factor (VEGF). Notably, <i>O</i>-xylosylation interactions with mannose receptor represent the first discovery highlighting potential immunomodulatory roles. <b>Conclusions</b>: This study highlights the critical importance of monitoring comprehensive glycosylation characterization during the development of BsAb with (G4S)n linkers to ensure optimal therapeutic efficacy, safety, and reduced immunogenic potential.