Comparison of free solution capillary electrophoresis and size exclusion chromatography for quantitating non-covalent...

There are few methods available for the rapid and precise quantitation of non-covalent aggregation. The very methods used to measure the aggregation can easily disrupt the weak forces holding an aggregate together. This paper describes the novel application of free solution capillary electrophoresis (CE) for the quantitation of a biologically inactive non-covalent aggregate of C8GLIP (Des-amino-histidine-7-arginine-26 N(epsilon)-octanoyl-lysine-34-human glucagon-like insulinotropic peptide), an acylated peptide. The CE results are compared to a more traditional approach using size exclusion chromatography (SEC). Under the conditions explored in this paper, SEC showed a significantly slower apparent rate of aggregation than CE. This is due to the disruption of the aggregate during the SEC process. The cause of the disruption is complex and is potentially related to the separation process itself, on-column dilution effects, and/or interactions of the aggregate with the column packing or SEC components. Analysis times and dilution are greatly reduced by CE, and, because there is no potentially interactive stationary phase and because both the protein and the walls of the capillary are negatively charged, potential disaggregation due to surface interactions is reduced. Thus, CE is shown to be superior to SEC for this peptide in that disruption of the aggregate is minimized.