Comparison of glycoprotein separation reveals greater impact of carbohydrates and disulfides on electrophoretic mobility for CE-SDS versus SDS-PAGE

Capillary electrophoresis sodium dodecyl sulfate (CE-SDS) has emerged as an indispensable tool in biopharmaceutical analysis to supersede the conventional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Despite the comparable migration behaviors of native proteins on the two platforms, the correlation has not been evaluated systematically for biotherapeutic proteins, which are often confounded by the presence of glycans and disulfides. Here we studied various mammalian glycoproteins using the two techniques under both reduced and non-reduced conditions. The results revealed substantial reduction in electrophoretic mobility under the capillary mode. Moreover, the migration order was found to be reversed between the reduced and nonreduced runs compared to SDS-PAGE. Such effects appeared to be independent of the content of sialylation. Our work unveiled complex interplays between the gel matrix, proteins and glycans which provide important guidance to biopharmaceutical analysis.