Fast separation and analysis of reduced monoclonal antibodies with capillary zone electrophoresis coupled to mass spectrometry.

Capillary zone electrophoresis–electrospray ionization–mass spectrometry (CZE–ESI–MS) was used for analysis of reduced antibodies. We first developed a simple protocol to condition commercial linear-polyacrylamide coated capillaries for use in top–down proteomics. We then suspended reduced antibodies in a solution of 35% acetic acid, 50% acetonitrile in water. Heavy and light chains were baseline resolved within 10 min and with 3–30 µg/mL detection limits using a 0.1% aqueous formic acid background electrolyte. Quintuplicate runs of a two-antibody mixture produced relative standard deviations of ∼1% in migration time and 10% in peak amplitudes. Resolution was further improved for the two-antibody mixture by using 5% acetic acid as the background electrolyte, highlighting the potential of capillary electrophoresis–mass spectrometry for analysis of antibody mixtures. [ABSTRACT FROM AUTHOR]