Characterization of bispecific and mispaired IgGs by native charge-variant mass spectrometry

Bispecific antibodies (BsAbs) combine the specificities of two antibodies to target different antigens. While production and in vivo assembly of BsAb using a single-cell host is less resource intensive compared to two-cell production, in particular mispaired light chain antibody species are a unique and undesired side product. Different pairing strategies may be employed to increase correct BsAb content, however, the potential presence of mispaired species must be both identified and quantified. Quantitative analysis by mass spectrometry is limited to chromatographic separation of the antibody assemblies due to their similar biophysical properties. This is particularly challenging for distinguishing correctly paired and light chain-scrambled mispaired antibodies which have the same molecular weight. Traditionally, imaged capillary isoelectric focusing (iCIEF) has been employed for analyzing charge-based heterogeneities, but this technique lacks resolution. Here we describe a powerful analytical platform using native weak cation exchange chromatography coupled to a high-resolution mass spectrometer, charge variant mass spectrometry (CV-MS), to characterize bispecific and mispaired antibody species. We investigate elution order through analytical methods and molecular modeling in an effort to understand the intrinsic charge, size and shape differences of these molecules. Although isoelectric points of the variants are similar, localized charge patches are hypothesized to offer sufficient micro-variations to be able to resolve and then identify isobars using CV-MS.