The Chromatography Forum

of Delaware Valley

Proteoforms are distinct molecular species of a protein that arise from a single gene by genetic variations, alternative splicing and post-translational modifications. Functional proteoformics or sorting proteoforms by conserved structural or functional features is enabling the discovery of new drugs and therapies. We describe here functional proteomics approaches for the analysis of quality attributes of recombinant therapeutic proteins and highlight their enabling features.

All the described technologies were performed in a flow steam with monoclonal antibodies (IgGs) as analytes. The Fc domain, a structural feature common to all mAb analytes, was specifically non-covalently labeled in a flow stream with a fluorescent affinity selector. Only mAb proteoforms containing a functional Fc domain quaternary structure were therefore analyzed without the need for mAb purification before analysis. We describe how these Fluorescent Amplified Structure Targeting (FAST) assays can be combined with LC fractionation of the analyte within the Proteometer platforms to deliver multiple quality metrics of mAbs in one run. These FAST Proteometer assays are highly reproducible. They enable rapid ranking of mAb samples based on quality attributes and performance metrics, thereby facilitating AI-driven decision-making.