Online top-down mass spectrometric identification of CE(SDS)-separated antibody fragments by two-dimensional...

Size heterogeneity analysis by capillary sieving electrophoresis utilizing sodium dodecyl sulfate (CE(SDS)) with optical detection is a major method applied for release and stability testing of monoclonal antibodies (mAbs) in biopharmaceutical applications. Identification of mAb-fragments and impurities observed with CE(SDS) is of outstanding importance for the assessment of critical quality attributes and development of the analytical control system. Mass spectrometric (MS) detection is a powerful tool for protein identification and characterization. Unfortunately, CE(SDS) is incompatible with online MS-hyphenation due to strong ionization suppression of SDS and other separation buffer components. Here, we present a comprehensive platform for full characterization of individual CE(SDS)-separated peaks by CE(SDS)-capillary zone electrophoresis-top-down-MS. The peak of interest is transferred from the first to the second dimension via an 8-port valve to remove MS-incompatible components. Full characterization of mAb byproducts is performed by intact mass determination and fragmentation by electron transfer dissociation, higher-energy collisional dissociation, and ultraviolet photodissociation. This enables online determination of intact mass as well as sequence verification of individual CE(SDS)-separated peaks simultaneously. A more substantiated characterization of unknown CE(SDS) peaks by exact localization of modifications without prior digestion is facilitated. High sensitivity is demonstrated by successful mass and sequence verification of low abundant, unknown CE(SDS) peaks from two stressed mAb samples. Good fragmentation coverages are obtained by MS², enabling unequivocal identification of these mAb-fragments. Also, the differentiation of reduced/non-reduced intra-protein disulfide bonds is demonstrated. In summary, a reliable and unambiguous online MS² identification of unknown compounds of low-abundant individual CE(SDS) peaks is enabled.