ELUTION BEHAVIOR OF SOLUBLE INTERCELLULAR-ADHESION MOLECULE-1 (SICAM-1) DURING SIZE EXCLUSION CHROMATOGRAPHY

A soluble recombinant form of intercellular adhesion molecule 1 (sICAM-1) has been analyzed by size exclusion high performance liquid chromatography (HPLC). In the course of the development of an analytical method utilizing this technique an anomalous chromatographic behavior was observed. In its native state, sICAM-1 was found to elute at a higher than expected molecular weight on columns calibrated with commonly used globular protein molecular weight markers. This was rationalized on the basis of known structural features of ICAM-1 as reported in the literature. Addition of sodium dodecylsulfate (SDS) to the mobile phase was found produce an improvement in the estimation of molecular weight. It is suggested that this improvement is a consequence of a tertiary structural rearrangement of the sICAM-1 molecule and, as such, provides further evidence to support the proposed native conformation. The anomalous size exclusion behavior and improvement in molecular weight estimation with SDS was confirmed on several different types of size exclusion columns. The study demonstrates the value of size exclusion HPLC for the rapid characterization of the overall shape of a recombinant protein of pharmaceutical interest and the utility of SDS therein.