Investigation of an artifact during non-reduced capillary electrophoresis with sodium dodecyl sulfate analysis utilizing N-ethylmaleimide as an alkylation reagent

This manuscript describes the formation of an artifact shoulder peak with a slightly larger retention time than the main peak under the standard non-reduced capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS) analysis of a therapeutic recombinant protein X, and clarifies the formation mechanism of the artifact caused by N-ethylmaleimide (NEM) during the sample preparation procedure. A design of experiment (DoE) approach was used to investigate the impact of the factors on the formation of the impurity. Additionally, orthogonal analytical experiments were performed to study the root cause of this phenomenon. The results consistently suggested that the Michael addition reaction between NEM and lysine residues in protein X, and decreased electrophoretic mobility due to increased molecular weight, was the root cause for the artifact, which could be partially inhibited by modifications of incubation conditions. Thus, before performing the nrCE-SDS method, the effects of alkyl-ation reagents and sample preparation procedure on analytical results need to be considered seriously.

Automated summary

This manuscript describes the formation mechanism of an artifact shoulder peak in a therapeutic recombinant protein X during the sample preparation procedure under standard non-reduced capillary electrophoresis with sodium dodecyl sulfate (nrCE-SDS) analysis. The results suggest that the reaction between NEM and lysine residues in the protein, as well as the decrease in electrophoretic mobility due to increased molecular weight, are the root causes for the formation of the artifact peak.