Journal of Chromatography A, 1238 (2012) 1–10 Contents lists available at SciVerse ScienceDirect Journal of Chromatography A jou rnal homepage: www.elsevier.com/locate/chroma Review Aqueous two-phase systems for protein separation: Phase separation and applicationsଝ ∗ Juan A. Asenjo, Barbara A. Andrews Centre for Biochemical Engineering and Biotechnology, Department of Chemical Engineering and Biotechnology, Institute for Cell Dynamics and Biotechnology: A Centre for Systems Biology, University of Chile, Santiago, Chile a r t i c l e i n f o a b s t r a c t Article history: Aqueous two-phase systems (ATPS) that are formed by mixing a polymer (usually polyethylene glycol, Received 31 January 2012 PEG) and a salt (e.g. phosphate, sulphate or citrate) or two polymers, and water can be effectively used Received in revised form 13 March 2012 for the separation and purification of proteins. The partitioning between both phases is dependent on the Accepted 15 March 2012 surface properties of the proteins and on the composition of the two phase system as has been recently Available online 23 March 2012 reviewed by Asenjo and Andrews [1]. This paper analyses and reviews some elements that are important for implementation of these processes which are related to phase separation and continuous processing Keywords: of ATPS. Phase separation for ATPS formed by PEG and salts has been studied and has been found to Aqueous two-phase systems depend on which of the phases is continuous. Profiles of dispersion heights can be represented as a Phase separation Applications fraction of the initial height and are independent of the dimensions of the separator. This is important for the design of large scale aqueous two-phase separations. The kinetics of phase separation has been investigated as a function of the physical properties of the system. The settling rate is a crucial parameter for equipment design and it has been studied as a function of viscosity and density of the phases as well as the interfacial tension between them. Correlations that describe the rate of phase separation have been developed. Working in a continuous bottom-phase region is advantageous to ensure fast separation. A mathematical model to describe the continuous, study state operation of these systems has been investigated. Two simulations to show the effect of phase ratio on purification have been carried out which clearly show the effectivity of using such models. The practical application of ATPS has been demonstrated in many cases including a number of industrial applications with excellent levels of purity and yield. Examples include the purification of ␣-amylase and the large scale “in situ” purification of IGF- 1 carried out by Genentech. The production scale purification of chymosin from recombinant Aspergillus supernatant is the most successful industrial application of this technology. Other applications include the separation and purification of human ␣-antitrypsin from transgenic sheep milk, the purification of monoclonal antibodies, tPA from CHO supernatant and recombinant VLPs (virus like particles) from yeast cells. © 2012 Elsevier B.V. All rights reserved. Contents 1. Introduction . 2 2. Phase formation and kinetics and phase separation . 2 3. Phase separation and kinetics . 3 4. Phase separation rates: correlation with system properties . 4 4.1. Effect of tie line length on the viscosity, density and interfacial tension between the phases . 4 4.2. Rate of separation; rate data correlation . 5 5. A mathematical model of aqueous two-phase continuous protein extraction . 6 5.1. Model of protein separation. 6 5.2. Mass balances . 6 ଝ Presented at the 16th International Conference on BioPartitioning and Purification, Puerto Vallarta, Jalisco, Mexico, 18–22 September 2011. ∗ Corresponding author. Tel.: +56 2 9784710; fax: +56 2 6991084. E-mail address: [email protected] (B.A. Andrews). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2012.03.049 2 J.A. Asenjo, B.A. Andrews / J. Chromatogr. A 1238 (2012) 1–10 5.3. Phase equilibrium . 6 5.4. Binodial and partition coefficient fitting . 6 5.5. Simulation examples: small PEG phase or large PEG phase . 7 6. Applications . 7 7. Conclusions . 9 Acknowledgement . 10 References . 10 1. Introduction Generally, the higher the molecular weight of the polymers the lower the concentration needed for the formation of two phases, Two phase aqueous partitioning is a very mild method of pro- and the larger the difference in molecular weights between the tein purification, and denaturation or loss of biological activity are polymers the more asymmetrical is the curve of the phase diagram not usually seen. This is probably due to the high water content and [3]. low interfacial tension of the systems which will protect the pro- During the last few years there has been renewed interest in teins. The polymers themselves may also have a stabilizing effect. separation methods for biological molecules as alternatives to chro- Aqueous two-phase partitioning can be used to separate proteins matography. Chromatography involves high costs, batch operation, from cell debris or to purify proteins from other proteins. Most sol- low throughput and complex scale-up [8]. Aqueous two-phase sys- uble and particulate matter will partition to the lower, more polar tems have recently been proposed for antibody purification [9–11]. phase, whilst proteins will partition to the top, less polar and more Rosa et al. [10] studied the economic and environmental sustain- hydrophobic phase, usually PEG [1–4]. Separation of proteins from ability of an ATPS process compared to protein A chromatography. one another is achieved by manipulating the partition coefficient by The ATPS process was found to be advantageous in terms of process altering the average molecular weight of the polymers, the type of economics when processing high titer cell supernatants. ions in the system, the ionic strength of the salt phase, by adding an additional salt such as NaCl or the presence of hydrophobic groups [5–7]. 2. Phase formation and kinetics and phase separation The partition coefficient (K) is defined as Why do systems with a continuous top phase take longer to sep- CT K = (1) arate than systems with a continuous bottom phase? It appears that CB the balance of forces on the drops during coalescence is different in where CT and CB represent the equilibrium concentrations of the each case. In general, three forces are acting on a drop during coa- partitioned protein in the top and bottom phases, respectively. lescence: gravitational, flotation and frictional, as shown in Fig. 1 The following properties of partitioning can be exploited indi- [12]. The movement of a drop depends on the balance between vidually or in conjunction to achieve an effective separation of a these forces. The gravitational force depends on the density of particular protein [1]. the drops, flotation or frictional forces depend on the rheologi- cal properties of the phases. The frictional force always impedes (i) Hydrophobicity, where the hydrophobic properties of a phase drop movement. These forces together with the interfacial tension, system are used for separation according to the hydrophobicity determine the coalescence behaviour and the characteristics of the of proteins. dispersed phase. In ATPS the densities of phases are very similar so (ii) Electrochemical, where the electrical potential between the it is the flotation forces which determine the behaviour of the drops. phases is used to separate molecules or particles according to The ratio of the viscosities of the polymer and the salt phase can be their charge. very large (5–50 times), the salt phase being much less viscous than (iii) Size-dependent partitioning where molecular size of the pro- the polymer phase. When the bottom phase is discontinuous, the teins or surface area of the molecules (proteins) or particles is coalescing drops must descend through the polymer phase. As the the dominating factor. polymer phase has a higher viscosity, the friction between the drops (iv) Biospecific affinity, where the affinity between sites on the pro- and the phase is high, hence the separation time is longer. When the teins and ligands attached to one of the phase polymers is bottom phase is continuous drops of the top phase move through exploited for separation. the bottom phase, which has a much lower viscosity, favouring (v) Conformation-dependent, where the conformation of the pro- coalescence. teins is the determining factor. An important element is which phase will be continuous and which the dispersed. Settling rates have been studied in PEG/salt Thus, the overall partition coefficient can be expressed in terms ATPS [12,13]. Given the high viscosity, rates are much smaller when of all these individual factors: the PEG top phase is the continuous phase. Phase separation times for polyethylene glycol (PEG)-4000-phosphate aqueous two-phase K = K · K · K · K · K · K 0 hfob el biosp size conf (2) systems have been studied [14], for small scale (5-g) and larger where hfob, el, biosp, size and conf stand for hydrophobic, electro- scale (1300-g) systems. Profiles of dispersion height for both larger chemical, biospecific, size and conformational contributions to the and small scale systems were represented as a fraction of the initial partition coefficient and K0 includes other factors. height and were found to be independent of the geometrical dimen- The factors which influence partitioning of a protein in aqueous sions of the separator. Furthermore, by plotting time as a fraction of two-phase systems are: the initial height the total time of separation can be calculated for a given height of system in a particular system as shown in Fig. 2a (i) molecular weights/size of polymers; for systems of two different sizes. This generalization is important (ii) concentration of polymer; for the design of large scale aqueous two-phase separators.