Comparison of free solution capillary electrophoresis and size exclusion chromatography for quantitating non-covalent aggregation of an acylated peptide

There are few methods available for the rapid and precise quantitation of n on-covalent aggregation. The very methods used to measure the aggregation c an 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 aggregat e of C8GLIP (Des-amino-histidine-7-arginine-26 N-epsilon-octanoyl-lysine-34 -human glucagon-like insulinotropic peptide), an acylated peptide. The CE r esults are compared to a more traditional approach using size exclusion chr omatography (SEC:). Under the conditions explored in this paper, SEC showed a significantly slower apparent rate of aggregation than CE. This is due t o 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 wi th the column packing or SEC components. Analysis times and dilution are gr eatly reduced by CE, and, because there is no potentially interactive stati onary phase and because both the protein and the walls of the capillary are negatively charged, potential disaggregation due to surface interactions i s reduced. Thus, CE is shown to be superior to SEC for this peptide in that disruption of the aggregate is minimized. (C) 1999 Elsevier Science B.V. A ll rights reserved.