Toward a screening method for the analysis of small intact proteins by CE‐ESI‐TOF MS

Abstract

Capillary electrophoresis-mass spectrometry (CE-MS) more and more gains in importance as an analytical technique for the identification and characterization of intact proteins in the biopharmaceutical area. Thus, a CE-ESI-MS method was optimized and validated systematically with respect to the improved screening and characterization of intact proteins. The optimization was accomplished by variation of different CE-MS parameters, such as capillary coating, background electrolyte, sheath liquid, and nebulizer gas pressure, while monitoring both the resolution and signal intensities. Achievable separation is discussed quantitatively in the context of the coating and the resulting EOF, the protein mobilities, and the suction effect of the sprayer. The observed precisions of the optimized method regarding the migration times (mean RSD = 1.4%) and peak areas (mean RSD = 12.3%) and an extensive principal component analysis revealed that the presented method is reliable and useful for the quantitation of intact proteins and protein isoforms. The applicability of this method to various proteins showing different characteristics (pI value, molecular mass, hydrophobicity, etc.) is discussed. The presented method will contribute to the improved characterization of a large variety of intact proteins in the biomedical and pharmaceutical area.