Fc Profiling of Polyclonal IgG, IgA and IgM by Light Chain Capturing Coupled with NanoRP-LC-MS

The proteoform profile of antibody Fc domains determines antibody effector functions, not only for biopharmaceuticals but also for endogenous antibodies. Endogenous immunoglobulin G (IgG) Fc-proteoforms have been well characterized by using different MS-based approaches, comprising bottom-up and intact Fc domain workflows. However, assessment of IgA1 and IgM Fc domains is still challenging, due to the more complex structure, and analyses have been limited to the peptide level only. In this work, a light-chain affinity capturing workflow combined with isotype-specific hinge-region digestion and subsequent intact Fc domain nanoRP-LC-MS analysis has been developed. The novel approach shows very good sensitivity and precision, enabling simultaneous capturing of antibody isotypes with sequential release and analysis of IgG, IgA1 and IgM Fcs from 10 μL of plasma. Single donor human samples were successfully analyzed, providing a comprehensive overview on Fc proteoforms but also on associated Fc-components such as the joining (J) chain of IgA and IgM and CD5L.