Protein refolding using size exclusion chromatography (SEC) is advantageous over conventional refolding methods in terms of ease of automation, simultaneous purification capabilities, and the non-adsorptive protein-matrix interaction which eliminates steric constraints. Despite these advantages, the widespread use of SEC refolding remains restricted by low process productivity and product concentration bottlenecks. This study aims to address those limitations and exploit SEC advantages for large-scale refolding applications. Specifically, this study reports the development of a pulsed-fed size exclusion chromatography (PF-SEC) refolding platform, which successfully refolded E. coli-derived alpha-fetoprotein (AFP) to achieve 53% refolding yield at 0.9 mg/ml AFP refolding concentration. AFP was introduced into the column by pulsed injection to increase feed load, while suppressing concentration-induced aggregation. Refolding was initiated by a urea gradient in the column, where the gradient length could be readily adjusted to complement pulsed feeding patterns. AFP refolding productivity with PF-SEC improved by 8- and 64-fold compared to ion-exchange chromatography refolding and pulsed dilution refolding, respectively, at a fixed refolding concentration. Through a unique integration of pulsed feeding and urea gradient development. this new PF-SEC refolding methodology overcomes 'productivity and concentration'-related disadvantages inherent in SEC, and will be scalable for large-scale protein refolding applications. (C) 2010 Elsevier Ltd. All rights reserved.