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.