Size exclusion HPLC method for the determination of acidic fibroblast growth factor in viscous formulations.
A size exclusion HPLC method has been developed to determine the protein concentration of pharmaceutical formulations of recombinant acidic fibroblast growth factor (aFGF). These topical aFGF formulations not only contain low levels of protein mass (50 micrograms ml-1), but also include buffer ions, polysaccharide polyanions to conformationally stabilize aFGF and 1% hydroxyethylcellulose to increase the solution's viscosity. A cesium chloride mobile phase is utilized during SEC-HPLC to dissociate aFGF from the pharmaceutical excipients and to minimize nonspecific interaction of the protein with the column matrix. The protein content of a viscous aFGF formulation is determined by comparison of aFGF peak areas to standards of known concentration. Fluorescence spectroscopy was utilized to directly demonstrate that the protein remains in its native conformation during sample preparation and analysis.
Bruner
M W
MW
Department of Vaccine Pharmaceutical Research, Merck Research Laboratories WP78-302, PA 19486, USA.
Goldstein
J
J
Middaugh
C R
CR
Brooks
M A
MA
Volkin
D B
DB
D002626:Chemistry, Pharmaceutical; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004355:Drug Stability; D016220:Fibroblast Growth Factor 1 / Q000032:analysis*; D007267:Injections; D016014:Linear Models; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis*; D015203:Reproducibility of Results; D014783:Viscosity
D011994:Recombinant Proteins; D016220:Fibroblast Growth Factor 1