Antibody-drug conjugates (ADCs) are used to deliver cytotoxic agents to tumor sites. These conjugates, which leak out from neovascular vessels via the enhanced permeability and retention (EPR) effect, bind to cell surface antigens and are internalized, thereby releasing their cytotoxic agents site-specifically. Conjugation sites and drug-to-antibody ratios influence the efficacy of ADCs; therefore, methods that allow homogeneous ADC preparation are required. While conjugation site and drug-to-antibody ratios affect the efficacy of ADCs, non-human type N-glycan structures are known to be immunogenic. We will solve these two problems by conjugating drugs via a conserved N-glycan at Asn297. The scheme is as follows: 1) Heterogeneous N-glycan on antibody was cleaved by endoS; 2) Human-type N-glycan from egg yolk was modified by azide addition and oxazoline formation: 3) The modified N-glycan was added to the antibody carrying enzymatically truncated N-glycan by endoS D233Q: 4) Drug carrying linker was conjugated via Huisgen reaction. Reaction progress was monitored by HILIC column chromatography without enzymatic digestion of the antibody. Using the above scheme, a homogeneous ADC was prepared. Homogeneity was quantitated by mass spectroscopy after proteolytic digestion of the modified antibody.
