Interference‐free mass spectrometric detection of capillary isoelectric focused proteins,including charge variants of a model monoclonal antibody

CIEF represents an elegant technique especially for the separation of structural similar analytes, whereas MS is a state‐of‐the‐art instrumentation for the identification and characterization of biomolecules. The combination of both techniques can be realized by hyphenating CIEF with CZE‐ESI‐MS applying a mechanical valve. During the CZE step, the remaining ESI‐interfering components of the CIEF electrolyte are separated from the analytes prior to MS detection. In this work, a multiple heart‐cut approach is presented expanding our previous single heart‐cut concept resulting in a dramatical reduction of analysis time. Moreover, different sample transfer loop volumes are systematically compared and discussed in regard to peak width and transfer efficiency. With this major enhancement, model proteins (1.63–9.75 mg/L), covering a wide pI range (5–10), and charge variants from a deglycosylated model antibody were analyzed on intact level. The promising CIEF‐CZE‐MS setup is expected to be applicable in different bioanalytical fields, e.g. for the fast and information rich characterization of therapeutic antibodies.