Aim: To demonstrate the capabilities of our new capillary electrophoresis –
mass spectrometry method, which facilitates highly accurate relative quantitation of
modification site occupancy of antibody-ligand (e.g., antibody-drug) conjugates.
Background: Antibody-drug conjugates play important roles in medical discovery for
imaging and therapeutic intervention. The localization and stoichiometry of the
conjugation can affect the orientation, selectivity, specificity, and strength of molecular
interactions, influencing biochemical function.
Objective: To demonstrate the option to analyze the localization and stoichiometry of
antibody-ligand conjugates by using essentially the same method at all levels including
ligand infusion, peptide mapping, as well as reduced and intact protein analysis.
Materials and Methods: Capillary electrophoresis coupled with electrospray ionization
mass spectrometry was used to analyze the antibody-ligand conjugates.
Results: We identified three prevalent ligand conjugation sites with estimated
stoichiometries of 73, 14, and 6% and an average ligand-antibody ratio of 1.37,
illustrating the capabilities of CE-ESI-MS for rapid and efficient characterization of
Conclusion: The developed multilevel analytical method offers a comprehensive way to
determine the localization and stoichiometry of antibody-drug conjugates for molecular
medicinal applications. In addition, a significant advantage of the reported approach is
the small, hydrophilic, unmodified peptides well separated from the neutrals, which is not
common with other liquid phase separation methods such as LC.