Previously we established a bench-scale system for the production of basic fibroblast growth factor (bFGF, FGF-2) from E. coli. In order to further identify and characterize this potent pharmaceutical protein, biologic activity tests, lyophilization tests, and long-term stability tests have been performed. Mitogenic activity of FGF-2 is tested by a dose-dependent cell proliferation assay using the fibroblast cell line NIH-3T3 and other cell lines, including embryonal stem cells and mesenchymal stem cells. FGF-2-stimulated neuronal differentiation on PC 12 cells is also tested. Lyophilization of FGF-2 is performed on a laboratory freezing dryer. The FGF-2 formulation aliquots are frozen at a speed of 20 C/h to ±25 C and then transferred to a ±80 C freezer overnight. Primary drying is performed at ±25 C to +10 C shelf temperature for 24 h, with <0.12 mbar vacuum level and ±60 C condenser temperature. The second drying is carried out at a shelf temperature of +25 C for 12 h with <0.06 mbar vacuum level and at the same condenser temperature as in the primary drying. The bioactivities before and after dehydration are compared. The stability of lyophilized FGF-2 is tested at multiple isothermal temperatures (±80 C, +4 C, +20 C, +37 C). Three stability-indicating methods (bioassay, SDS-PAGE, RP-HPLC) are used to evaluate the long-term stability of this formulation. The data are fit into the Arrhenius equation and the shelf-life of lyophilized protein is predicted by the Arrhenius plot. P7.82Gewinnung

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