Aims:
Demonstrating 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 to electrospray ionization mass spectrometry was used to analyze the antibodyligand 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 antibody-drug conjugates.
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 that small, hydrophilic, unmodified peptides well
separated from the neutrals, which is not common with other liquid phase separation methods such as LC.
Application of triple quadrupole mass spectrometry for the characterization of antibody–drug conjugates
