SIZE-EXCLUSION HPLC METHOD FOR THE DETERMINATION OF ACIDIC FIBROBLASTGROWTH-FACTOR IN VISCOUS FORMULATIONS

A size exclusion HPLC method has been developed to determine the prote in concentration of pharmaceutical formulations of recombinant acidic fibroblast growth factor (aFGF). These topical aFGF formulations not o nly contain low levels of protein mass (50 mu g ml(-1)), but also incl ude buffer ions, polysaccharide polyanions to conformationally stabili ze aFGF and 1% hydroxyethylcellulose to increase the solution's viscos ity. A cesium chloride mobile phase is utilized during SEC-HPLC to dis sociate aFGF from the pharmaceutical excipients and to minimize nonspe cific interaction of the protein with the column matrix. The protein c ontent of a viscous aFGF formulation is determined by comparison of aF GF peak areas to standards of known concentration. Fluorescence spectr oscopy was utilized to directly demonstrate that the protein remains i n its native conformation during sample preparation and analysis. (C) 1997 Elsevier Science B.V.