ISSN:
1573-904X
Keywords:
fibroblast growth factor
;
protein stability
;
formulation
;
HPLC
;
denaturation kinetics
;
fluorescence
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
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. Re versed-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.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1018946011652
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