Development of Monolithic Silica Capillary Columns for LC/MS Analysis of Intact Proteins

Abstract

A monolithic silica capillary column suitable for intact protein separation was developed for the high-resolution separation of proteins contained in biological samples by LC or LC/MS analysis. While investigating of the stationary phases, monolithic silica capillary columns modified with C4 or phenyl groups showed less carryover for high-molecular-weight monoclonal antibodies than C8 modification. Furthermore C4-modified monolithic silica capillary columns with lower adsorption are suitable for protein analysis containing unknown and low concentration proteins. In addition, we can evaluate the thermal degradation and denaturation of proteins using monoclonal antibodies, since a C4-modified monolithic silica capillary column can perform LC separation of proteins at relatively low temperature. As a result, it was suggested that it was important to keep the column temperature below 60 °C for the quantitative analysis of proteins. The C4-modified monolithic silica capillary column is useful for intact protein separation analysis, such as high-speed LC/MS analysis of monoclonal antibodies and high-resolution separation analysis of human cell-derived proteins.