Systematic characterization of lysine glucuronidation in a bispecific antibody

This study presents a systematic characterization of lysine glucuronidation that was revealed during the charge variant characterization of a bispecific antibody (bsAb). Site-specific quantitation by Glu-C/Asp-N peptide mapping suggested that glucuronidation occurred randomly across surface lysine residues. To understand the impact of glucuronidation on the structure and function of the bsAb, stressed samples with up to 84% total glucuronidation were generated and analyzed by a comprehensive panel of analytical methods. The results suggested that glucuronidation caused an acidic isoelectric point (pI) shift in the charge profile. However, it does not affect the higher-order structure or bioactivities of the bsAb, including antibody-dependent cell-mediated cytotoxicity, antigen binding, or Fc receptor interaction. To support routine process monitoring, a fit-for-purpose subunit mass method was developed and qualified for quantitation of glucuronidation, offering a higher-throughput alternative to peptide mapping for assessing process consistency and product comparability.