Recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed as cytoplasmic inclusion bodies (IBs) in Escherichia coli, was recovered with higher yield and purity by developing an optimized process involving the sequential evaluation of complete downstream processes. Triton X-100 and urea wash removed 90% of lipopolysaccharides, 22% of host DNA and 98% of host cell proteins, leading to inclusion bodies purity of 99% with 91% recovery. Removal of host impurities at solubilization step and refolding by diafiltration at high concentration of protein were identified as key parameters with significant impact on product recovery and purity. Desalting, size exclusion and formulation were performed in a single step. This optimized process recovery yielded 69% (34 mg from 1 g of wet cell mass) of purified protein with more than 99% purity. According to available literature, the purified rhG-CSF yield obtained in this study is the highest that has been reported so far. (C) 2008 Elsevier Ltd. All rights reserved.