Direct Comparison of the Hinge-Cleaving Proteases IgdE and BdpK for LC-MS-Based IgG1 Clonal Profiling

Abstract

Human antibodies are heterogeneous molecules primarily due to clonal sequence variations. Analytical techniques to assess antibody levels quantitatively, such as ELISA, lack the power to resolve abundances at the clonal level. Recently, we introduced an LC-MSbased approach that can distinguish and quantify antibody clones using the mass and retention time of their corresponding Fab-fragments. We used specific hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments from the constant Fc region of the antibody. Here, we explore an alternative IgG1 hinge-cleaving protease, BdpK (FabDELLO), and compare it directly to IgdE for use in IgG1 repertoire profiling. We used IgdE and BdpK in parallel to digest all IgG1s from the same set of plasma samples. Both proteases cleave IgG1 specifically in the hinge, albeit via different mechanisms and at two distinct cleavage sites. Notwithstanding these differences, the Fab fragments generated by IgdE or BdpK produced highly similar clonal repertoires. However, IgdE required ∼16 h of incubation to digest plasma IgG1s, while BdpK required ∼2 h. We authenticated the similarity of the clones by top-down proteomics using electron transfer dissociation. We conclude that BdpK performs very well in digesting polyclonal plasma IgG1s and that neither BdpK nor IgdE displays detectable biases in cleaving IgG1s. We anticipate that BdpK may emerge as the preferred protease for IgG1 hinge-digestion because it offers a shorter digestion time compared to IgdE, an equally specific digestion site, and no bias against any IgG1 present in plasma.

Protein-Centric Analysis of Personalized Antibody Repertoires Using LC-MS-Based Fab-Profiling on a timsTOF

“…In our initial studies on antibody repertoires, using Orbitrap-based platforms and microflow LC, we injected sample amounts corresponding to 2–4 μL of plasma or serum per injection and could then typically identify ∼250–550 distinct Fab clones . The current study demonstrates that moving toward nanoLC-MS scale has potential benefits such as lower sample consumption, better separation efficiency, improved ionization efficiency, and could therefore lead to deeper insight into antibody repertoires .…”