This work reports the upstream and downstream process of recombinant human granulocyte colony stimulating factor (rhG-CSF) expressed in Escherichia coil BL21 (DE3)pLysS. The fed batch mode was selected for the maximum output of biomass (6.4 g/L) and purified rhG-CSF (136 mg/L) under suitable physicochemical environment. The downstream processing steps viz., recovery, solubilization, refolding and concentration were optimized in this study. The maximum rhG-CSF inclusion bodies recovery yield (97%) was accomplished with frequent homogenization and sonication procedure. An efficient solubilization (96%) of rhG-CSF inclusion bodies were observed with 8 M urea at pH 9.5. Refolding efficiency studies showed maximum refolding >= 86% and >= 84% at 20 degrees C and pH 9 respectively. The renatured protein solution was concentrated, clarified and partially purified (>= 95%) by the cross flow filtration technique. The concentrated protein was further purified by a single step size exclusion chromatography with >98% purity. The characterization of purified rhG-CSF molecular mass as evidenced by SDS-PAGE, western blot and LC/MS analysis was shown to be 18.8 kDa. The secondary structure of rhG-CSF was evaluated by the CD spectroscopic technique based on the helical structural components. The biological activity of the purified rhG-CSF showed a similar activity of cell proliferation with the standard rhG-CSF. Overall, the results demonstrate an optimized downstream process for obtaining high yields of biologically active rhG-CSF. (C) 2015 Elsevier Inc. All rights reserved.