This work describes the integration of expanded bed adsorption (EBA) and adsorptive protein refolding operations used to recover purified and biologically active human basic fibroblast growth factor from inclusion bodies expressed in E. coli. Insoluble overexpressed human basic fibroblast growth factor has been purified on CM Hyper Z matrix by expanded bed adsorption after isolation and solubilization in 8？M urea. The adsorption was made in expanded bed without clarification steps such as centrifugation. Column refolding was done by elimination of urea and elution with NaCl. The human basic fibroblast growth factor was obtained as a highly purified soluble monomer form with similar behavior in circular dichroism and fluorescence spectroscopy as native protein. A total of 92.52% of the available human basic fibroblast growth factor was recovered as biologically active and purified protein using the mentioned purification and refolding process. This resulted in the first procedure describing high-throughput purification and refolding of human basic fibroblast growth factor in one step and is likely to have the greatest benefit for proteins that tend to aggregate when refolded by dilution. 1. Introduction Production of therapeutic proteins in inclusion bodies is useful due to the efficacy of insoluble expression such as high product yield and protection against degradation by proteases [1]. The recovery of biologically active protein from such inclusion bodies requires refolding protocols. In general, the methods used for inclusion body solubilization result in a soluble protein that is biologically inactive. The solublized proteins do not have native conformation and must be transferred into conditions that allow the formation of the native structure. During this period, the correct folding pathway competes, often in disadvantage with misfolding and aggregation of the target protein. Protein refolding involves intramolecular interactions and follows first-order kinetics [2]. Refolding yields commonly decrease with increasing initial concentrations of the unfolded protein independent of the refolding method applied [3]. Aggregates are formed by nonnative intramolecular hydrophobic interactions between protein folding intermediates, which have not yet buried their hydrophobic amino acid stretches. Therefore, prevention of hydrophobic intermolecular interaction during the first steps of refolding is crucial to allow successful renaturation at high protein concentrations [4]. A very efficient strategy to prevent aggregation is to minimize the risk of
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