BIOSEPARATIONS IN AQUEOUS MICELLAR SYSTEMS BASED ON EXCLUDED-VOLUME INTERACTIONS

0960–3085/06/$30.00+0.00

# 2006 Institution of Chemical Engineers

www.icheme.org/fbp Trans IChemE, Part C, March 2006

doi: 10.1205/fbp.05159 Food and Bioproducts Processing, 84(C1): 51 – 58

BIOSEPARATIONS IN AQUEOUS MICELLAR SYSTEMS BASED ON EXCLUDED-VOLUME INTERACTIONS

D. VAN ROOSMALEN1, M. P. J. DOHMEN-SPEELMANS1, C. H. J. T. DIETZ1, L. J. P. VAN DEN BROEKE1 , L. A. M. VAN DER WIELEN2 and J. T. F. KEURENTJES1

1Process Development Group, Department of Chemical Engineering and Chemistry, Eindhoven University of Technology, Eindhoven, The Netherlands

2Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

P rotein partitioning based on micellar-induced excluded-volume interactions is studied for different aqueous nonionic micellar systems. In particular, protein partitioning in the traditional aqueous micellar two-phase system is compared with a system where

a gel phase is combined with a micellar phase and with a membrane system with micelles at the feed side of the membrane. A two-phase system is created using cylindrically-shaped n-decyl tetra(ethylene oxide) (C10E4) micelles that are too large to diffuse into the gel-bead or across the membrane. Results are reported for the partition coefficient of single-component protein systems with myoglobin, ovalbumin, and BSA, and for binary protein mixtures. The individual protein partition coefficients in the mixtures follow the same protein concentration dependence as the partition coefficient obtained for the single-component protein solutions.

Keywords: nonionic surfactant; excluded-volume interactions; protein; partitioning; membrane; sephacryl gel.

INTRODUCTION

Separation and purification of biomolecules makes up the major part of the production costs for biological products (Goetheer et al., 1999; Cunha and Aires-Barros, 2002). Considering the range of biomolecules to be separated, there is a clear need for more flexible, and potentially more efficient and cost-effective, large-scale bioseparation techniques (Lightfoot and Moscariello, 2004; Noble and Agrawal, 2005). Typically, similar species in dilute streams have to be separated, like monomer – multimer protein sep-aration, and this should potentially include viral clearance (Aranha, 2001). Also in the analytical field, advanced new developments have been shown for the separation of protein-polypeptide mixtures (Manabe, 2003).

For the separation of biomolecules a variety of interactions and mechanisms is available. The main interactions include excluded-volume, hydrophobic, charge (or ion-exchange), and affinity or ligand interactions (Aboul-Enein, 1999). A convenient classification of separation systems is based on the mechanism of separation (Seader and Henley, 1998). For the separation of biomolecules, the use of a barrier

Correspondence to: Dr L. J. P. van den Broeke, Process Development

Group (SPD), Department of Chemical Engineering and Chemistry, Eind-

hoven University of Technology, PO Box 513, 5600 MB Eindhoven, The

Netherlands.

E-mail: l.j.p.van.den.broeke@tue.nl

or solid agent is the most wide spread (Lightfoot and Moscar-iello, 2004). This includes size exclusion chromatography (Barth et al., 1998; Wu, 1999), hollow fibres (Gabelman and Hwang, 1999) and membranes (Pujar and Zydney, 1998; Persson et al., 2003), integrative membrane chromato-graphy (Tho¨mmes and Kula, 1995; Ghosh, 2002), adsorption (Burton and Harding, 2001), micellar liquid chromatography (Berthod and Garcı´a-Alvarez-Coque, 2000; Baltus et al., 2002), and surfactant-aided size exclusion chromatography (Horneman et al., 2004).

Another mechanism of separation involves phase addition or phase creation. This includes precipitation (Tessier and Lenhoff, 2003; Lenhoff, 2003) and extraction. In particular, micelle-mediated extraction (Pires et al., 1996), such as liquid – liquid extraction using reverse micelles (Quina and Hinze, 1999; Tani et al., 1997) and aqueous two-phase-systems (Walter et al., 1991; Liu et al., 1998), is used for protein separation. Excluded-volume interactions are one of the main interactions used to partition proteins in aqueous nonionic micellar solutions (Nikas et al., 1992; Lazzara et al., 2000; White and Deen, 2001) and size-exclusion chromatography (Albertsson, 1956; Barth et al., 1998; Wu, 1999).

Micellar-Based Bioseparation

In this work, a generalized concept to use aqueous micellar two-phase systems for the partitioning of proteins

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is presented. The main mechanism for the partitioning is based on excluded-volume interactions, which are induced by the micellar phase. The idea is to construct two separate phases, i.e., a size-selective micellar phase and an aqueous micelle-poor phase, based on surfactants forming cylindri-cally-shaped micelles. This means that if a solid fibrous phase is used, like a gel or a membrane, with pore dimen-sions smaller than the length of the micelles, the micelles will be excluded from the solid phase and in this way the two separate phases are formed. Clearly, the pore size of the solid fibrous structure should be large enough to accommodate the proteins.

In Figure 1 the principle of constructing different two-phase configurations is schematically depicted. In order to obtain a size-selective micellar phase, referred to as phase I, which is physically separated from an aqueous phase, phase II, different methods for phase separation

(M) are applied. The methods of phase separation and the resulting two-phase systems, used in this work, are summarized in Table 1.

In the aqueous micellar two-phase system (AMTPS) there is no actual physical barrier between the micelle-rich and

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