Journal of Chromatography A, 1217 (2010) 7265–7274 Contents lists available at ScienceDirect Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma Ion-exchange chromatographic protein refolding Esteban J. Freydell a , Luuk van der Wielen a , Michel Eppink b,1 , Marcel Ottens a,∗ a Department of Biotechnology, Delft University of Technology, Delft, The Netherlands b Biotechnology Operations, N.V. Organon, Schering-Plough, Oss, The Netherlands article info Article history: Received 18 June 2010 Received in revised form 3 September 2010 Accepted 14 September 2010 Available online 19 September 2010 Keywords: Ion-exchange chromatography Protein refolding Fractional surface coverage Matrix assisted refolding abstract The application of ion-exchange (IEX) chromatography to protein refolding (IExR) has been successfully proven, as supported by various studies using different model proteins, ion-exchange media and flow configurations. Ion-exchange refolding offers a relatively high degree of process intensification, repre- sented by the possibility of performing protein refolding, product purification and product concentration, in one unit operation. Besides its high degree of process intensification, IExR offers an additional set of key advantages including: spatial isolation of the bound protein molecules and the controllable change in chemical composition using gradients. Despite of the acknowledgement of the former advantages, the lack of mechanistic understanding on how they influence the process performance of the ion-exchange refolding reactor, limits the ability to exploit them in order to optimize the performance of the unit. This paper presents a quantitative analysis that assesses the effect that the spatial isolation and the urea gradient, have on the IExR performance, judged on the basis of the refolding yield (Y N ) and the fractional mass recovery (f Prot,Rec ). Additionally, this work discusses the effect of the protein load, the protein load- ing state (i.e., native, denatured, denatured and reduced (D&R)) and the adsorbent type on f Prot,Rec . The presented work shows: (1) that the protein load has a direct effect on f Prot,Rec , and the magnitude of this effect depends on the loading state of the protein solution and the adsorbent type; (2) that irrespectively of the type of adsorbent used, the saturation capacity of a denatured protein is less than the native protein and that this difference can be linked to differences in accessible binding surface area; (3) that there is a clear correlation between fractional surface coverage () and f Prot,Rec , indicating that the former could serve as a good descriptor to assess spatial isolation, and (4) that the urea gradient has a direct link with the variations on the refolding yield, and this link can be quantitatively estimated using as descriptor the urea gradient slope (). Overall, the information provided in this paper aims at the eventual develop- ment of rational design or selection strategies of ion-exchange media for the satisfactory and successful refolding of a target protein. © 2010 Elsevier B.V. All rights reserved. 1. Introduction Developments attained in recombinant DNA technology have significantly changed the way valuable proteins are produced today. Proteins that used to be purified from human fluids, ani- mal or plant tissue can now be produced in large quantities using for example Escherichia coli (E. coli). As an expression system E. coli offers several advantages including (1) its molecular genetics are well understood, meaning that its genome can be modified with ease; (2) relatively inexpensive culturing procedures and (3) high fermentation yields [1–3]. One important limitation of this expres- sion system though, is that the over expression of certain gene sequences leads to the accumulation of the product in an inac- ∗ Corresponding author. Tel.: +31 15 2782151; fax: +31 15 2782355. E-mail address: m.ottens@tudelft.nl (M. Ottens). 1 Current address: Synthon N.V., Nijmegen, The Netherlands. tive, insoluble aggregate known as inclusion body (IB). To obtain the soluble and bio-active (native) product, two process steps are required and these are (1) inclusion bodies (IBs) solubilization and (2) protein refolding. IBs solubilization is usually done using a solution containing chaotropes (e.g., urea, guanidine hydrochlo- ride), reducing agents (e.g., dithiothreitol, -mercaptoethanol, etc.) and alkaline pH. This cocktail disrupts the intermolecular interac- tions holding the aggregated protein, releasing the product in a denatured and reduced (D&R) soluble form. Optimal solubilization conditions should provide maximum protein solubility, minimiz- ing the fraction of soluble aggregates formed and maximizing the fraction of soluble denatured and reduced monomer product [4–7]. Protein refolding is achieved by decreasing the concentra- tion of chaotropes and reducing agents in the concentrated protein solution, allowing the soluble protein to refold and to form its disul- phide bonds. This change in chemical composition is basically a buffer exchange step that can either be attained by direct dilution or using liquid chromatography (chromatographic refolding). 0021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2010.09.044