Ultrafast Microdroplet Digestion of Antibodies with Fc-Silencing Mutations

Many novel therapeutic monoclonal antibody (mAb) modalities contain mutations that not only silence unwanted binding to Fc-gamma receptors but also could bring resistance to IdeS enzymatic cleavage into mAb subunits, limiting the antibody middle-down analysis by mass spectrometry (MS). Herein we showed, for the first time, the efficient, reproducible, and ultrafast microdroplet digestion (less than 1 ms) of mAbs carrying a series of mutations "LALA", "LAGA", and "LFLE" (e.g., tool antibody LALA-DS, Nivolumab, Pembrolizumab, Vedolizumab, and PD-L1), by a new enzyme: FabRICATOR Xtra (Xtra). The digestion took place during the spray ionization process using an Agilent jet stream (AJS) ion source with a digestion efficiency close to or more than 80%, leading to subunits with high ion abundances for identification and characterization. Increased enzyme/antibody ratio or partial reduction of disulfide bonds increased the digestion efficiency. "One-pot" disulfide reduction and digestion in microdroplets could occur simultaneously by spraying the antibody and enzyme along with the reductant. Notably, for the highly digestion-resistant PD-L1 antibody, the Xtra-based microdroplet digestion process was found to be 9 million times faster than in-solution digestion. Furthermore, a workflow was developed, using a script that can automatically choose a preferred enzyme (Xtra or IdeS) for digestion, based on a target antibody input sequence, which allowed quick analysis of 94 antibody samples within 104 min. Our method is a fully automated microdroplet protein digestion technique that integrates flow injection (FI) and online MS analysis, providing a rapid and robust method for the structural characterization of mAbs with mutations.