Direct measurement and reconstruction of intact polyclonal IgG repertoires to preserve molecular and functional connectivity

Antibody function depends on the molecular pairing of antigen-binding Fab regions with Fc domains, linking antigen specificity to effector potential. However, antibody repertoire methods analyze these subunits separately, creating an inference problem in which Fc-Fab connectivity is lost. Here, we introduce a multi-level HPLC-MS workflow integrating native nanoflow cation-exchange chromatography-MS (nCEC-MS) of intact serum IgG with middle-up Fab and Fc/2 profiling. Native nCEC-MS reduces spectral congestion compared with denaturing reversed-phase HPLC-MS, enabling detection of 56-74 intact IgG mass features from 2 μg of purified IgG per donor. Middle-up analysis provided complementary information on Fab diversity, subclass, and allotype, revealing donor-specific profiles. Reconstructing intact IgG1 masses from independently measured Fab and Fc/2 subunits partially recovers Fc-Fab molecular connectivity and supports interpretation of native intact mass features. Together, this framework provides an integrated view of the human IgG repertoire and establishes a foundation for studying its molecular topology in health and disease.