Enhancement of covalent aggregate quantification of protein therapeutics by non-reducing capillary gel electrophoresis using sodium hexadecyl sulfate (CE-SHS).

Capillary gel electrophoresis (CGE) using sodium dodecyl sulfate (CGE-SDS or CE-SDS) is commonly used in the biotechnology industry to assess the purity of a complex therapeutic during manufacturing process optimization and also for commercial release and stability testing. However, for therapeutic proteins mAb-1 and mAb-2, non-reducing (NR) CE-SDS yielded higher than expected % aggregate which considerably lowered its apparent purity relative to the purity reported by other complementary methods, such as Size Exclusion Chromatography (SEC). Furthermore, a strong protein load dependence on aggregate levels was observed which prevented any reasonable assessment of the true purity value. The solution was to supplement SDS with the relatively hydrophobic detergent sodium hexadecyl sulfate (SHS) in the sieving gel buffer matrix which virtually eliminated the protein load-dependence and reduced the % aggregate value to expected levels when compared to SEC. Analytical Ultracentrifugation (AUC) was used to help confirm the accuracy of the SEC results. This work underscored how using detergents other than SDS in CGE applications can be valuable in the commercial biologics space and provided an example of how SEC can be used to confirm the accuracy of CGE data.