title: Chromatographic methods for quantitative analysis of native, denatured, and aggregated basic fibroblast growth factor in solution formulations.
abstract: High-performance liquid chromatography (HPLC) methods were developed for evaluating stability of human recombinant basic fibroblast growth factor (bFGF) against denaturation and aggregation in solution formulations. Reversed-phase chromatography (RP-HPLC)-insensitive to bFGF tertiary structure--was used to measure total soluble protein; heparin affinity chromatography (HepTSK) provided quantitative analysis of native bFGF species. The folding state of bFGF was determined by fluorescence spectroscopy: Tryptophan emission, which was quenched in native protein, increased upon unfolding. Slow unfolding/refolding kinetics of bFGF in 2 M guanidine hydrochloride made possible the separation of native from denatured species by size exclusion chromatography (SEC). Although the tertiary structure affected bFGF retention times, it did not change the sample recovery by SEC. These chromatographic techniques, which quantitatively measure physical and chemical changes taking place in solution formulations, can be used in future investigations of bFGF stability.
authors.affiliation: Pharmaceutical R & D, Scios Nova Inc., Mountain View, California.
authors.forename: V
authors.initials: V
authors.lastname: Sluzky
authors.forename: Z
authors.initials: Z
authors.lastname: Shahrokh
authors.forename: P
authors.initials: P
authors.lastname: Stratton
authors.forename: G
authors.initials: G
authors.lastname: Eberlein
authors.forename: Y J
authors.initials: YJ
authors.lastname: Wang
mesh_terms: D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D016222:Fibroblast Growth Factor 2 / Q000008:administration & dosage / Q000032:analysis*; D006493:Heparin / Q000737:chemistry; D006801:Humans; D007202:Indicators and Reagents; D008970:Molecular Weight; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000008:administration & dosage / Q000032:analysis; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
chemical_list: D007202:Indicators and Reagents; D011994:Recombinant Proteins; D012996:Solutions; D016222:Fibroblast Growth Factor 2; D006493:Heparin