0960–3085/02/$10.00+0.00 # Institution of Chemical Engineers Trans IChemE, Vol 80, Part C, March 2002 BIOPROCESS INTENSIFICATION: A RADICAL NEW PROCESS FOR RECOVERING INCLUSION BODY PROTEIN W. CHOE, R. CLEMMITT, M. RITO-PALOMARES, H. A. CHASE and A. P. J. MIDDELBERG Department of Chemical Engineering, University of Cambridge, Cambridge, UK T he successful development of a greatly simplied purication process for recombinant protein is described. The direct chemical extraction of recombinant L1 protein (the major capsid protein of human papillomavirus type 16) from cytoplasmic inclusion bodies in E. coli was demonstrated at high cell density (to OD 600 = 80). Highly efcient and selective precipitation of DNA was achieved during extraction using spermine, allowing direct coupling to an immobilized metal afnity column operated in expanded bed mode (IMAC–EBA). Direct capture of the target L1 protein from the extraction broth gave an unoptimized yield of 60% with a purication factor of 10. The demonstrated process greatly simplies the way in which protein expressed as an inclusion body can be recovered at process scale. Specically, the new process eliminates the need for mechanical cell disruption and inclusion body centrifugation, and direct EBA capture yields partially puried protein with only two units: fermentation and expanded bed adsorption. Keywords: chemical extraction; DNA precipitation; spermine; expanded bed adsorption; inclusion body INTRODUCTION With the increasing shift from genomics to proteomics there is a need to rapidly and economically bring new biophar- maceutical products to market using scaleable and efcient bioprocess technology. The large number of biopharmaceu- ticals approaching the end of their patent protection period has also encouraged manufacturers to seek competitive advantage through bioprocess intensication. In both cases, E. coli is often the rst host of choice, due mainly to the accumulated knowledge base regarding its molecular biology and metabolism 1 , and the high yields typically obtained by fed-batch fermentation 2 . However, an inclusion body often forms when recombinant protein is over- expressed in E. coli , and this contains predominantly the recombinant protein of interest in an inactive form, with some other contaminants. The protein must therefore be processed to its native state for biological activity 3,4 . A typical process route for the recovery of protein expressed as an inclusion body involves release of the inclusion bodies by high-pressure homogenization and their collection by centrifugation. Inclusion bodies are then solubilized in a strong denaturant prior to refolding through removal of denaturant. The entire process is compli- cated by the need for multiple cell-disruption passes to reduce cell-debris size 5 , and by the need to wash this debris from the inclusion bodies by repeated centrifugation 6 , possibly with the use of detergents and other chemical agents. The multi-step nature of this conventional method, together with the complexity of separating solids of similar particle size, can result in low process yield and high process cost. The large amount of stainless steel involved is also viewed as a signicant disadvantage. Consequently, inclusion bodies are perceived as an undesirable outcome of expression, despite the fact that their formation is usually associated with high expression yield and with protection of the protein from in vivo proteolysis. To overcome some of the disadvantages attributed to established methods for processing inclusion bodies, differ- ent approaches have been proposed. The use of membranes, in place of batch centrifugation for inclusion body collection and washing, resulted in a three-fold increase in yield 7,8 . However, a comparison with a more effective disc-stack centrifuge (for cell debris and inclusion body fractionation) was not reported. A signicant increase in the efciency of cellular disruption following the addition of detergent and chaotrope has been reported by Bailey et al. 9 , but the addition of detergent complicates subsequent processing without eliminating the mechanical disruption step. The use of aqueous two-phase systems (ATPS) for direct recov- ery of inclusion bodies following mechanical disruption has recently been reported 10 . However, inclusion bodies collect at the interface thus complicating their recovery and further processing. A different approach involving the in situ dissolution of periplasmic inclusion bodies with subsequent recovery of the soluble protein by ATPS has been developed by Hart et al. 11,12 . One of the present authors has extended this extraction method to achieve the in situ dissolution of cytoplasmic inclusion bodies using chaotrope (urea) and EDTA 13–15 . This new chemical extraction method 45