Liquid-liquid extraction of biomolecules in downstream processing - A review paper

Liquid-liquid extraction of biomolecules in downstream processing - a review paper

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Faculdade de Engenharia de Alimentos, Unicamp, Departamento de Tecnologia de Alimentos,

C.P. 6066, CEP 13081-970, Campinas - SP, Brasil

Faculdade de Engenharia Química, Unicamp, Departamento de Processos Químicos,

C.P. 6066, CEP 13081-970, Phone: [phone], Campinas - SP, Brasil

E-mail: [e-mail]

(Received: May 24, 1999; Accepted: July 27, 1999)

Abstract - Economic analysis shows that protein separation and purification are a very important aspect of biomolecules production and processing. This is particularly true for protein processing which, because of the complexity of the starting material, often requires many steps to reach the levels of purity required for medical and food applications. The separation specialists' task is to develop safe and simple processes to achieve products with a high level of purity. On a large scale, chromatography of proteins is not an easily applied method, although on a laboratory scale it is very effective and relatively simple. When it is scaled up, shortcomings such as discontinuity in the process, slow protein diffusion and large pressure drops in the system are seen. For these reasons a substantial research effort has been directed toward the use of aqueous two-phase systems (ATPSs) to replace the initial steps in protein purification and chromatography. This article reviews the chronology and main ATPS fundamentals and discuss the broader applications of this type of system in the extraction and separation of biomolecules.

Keywords: aqueous two-phase systems, downstream processing, protein purification, liquid-liquid extraction.

INTRODUCTION

AQUEOUS TWO-PHASE SYSTEMS

ATPS can easily be scaled up without an appreciable change in the nature or efficiency of the process. In addition, since there is no solid phase, thorough mixing of the two phases is possible and hence interphase transport is rapid. Only seconds are required to bring most two-phase systems into equilibrium. Another benefit is that the phases are compatible with almost all known proteins. They are an attractive alternative procedure for the separation and purification of proteins on a large scale. The question of selectivity in protein partitioning still needs to be better understood. An increased knowledge of protein behaviour in aqueous-two phase systems will also lead to the ability to predict the partitioning of target proteins, often found in a heterogeneous and complex mixture of proteins.

Partitioning in aqueous two-phase systems is mainly a process in which the exposed groups of molecules come into contact with the phase components, and it is therefore a surface-dependent phenomenon. The influence of different factors upon the partitioning of proteins is illustrated by the logarithm of the partition coefficient. This can be considered to be the sum of the logarithms of several terms:

lnK= lnKelec +lnKhphob +lnKbiosp +lnKsize +lnKconf

This equation includes a hydrophobic term (K_hphob), a term that includes electrostatic effects (K_elec) mainly determined by the protein net charge and hence pH, ion distribution and also the charge of the polymers involved. Protein and polymer size (K_size), conformation (K_conf) and affinity ligands (K_biosp) can also be important.

The tendency for two different aqueous polymers (e.g., gelatine and agar, gelatin and soluble starch) to separate into two distinct phases in a common solvent has been recognized since the end of last century, and it has been shown to be the general rule for most water-soluble polymer-polymer systems. For example, a mixture of dextran and polyethylene glycol dissolved in water is turbid above certain polymer concentrations, and the two phases are in equilibrium. The lighter phase is enriched in polyethylene glycol while the heavier is enriched in dextran (Figure 1). Both polymers are fully water soluble, yet the two polymers are incompatible and separate into two phases (Albertsson, 1986). Albertsson (1971) compared the two-phase aqueous polymer with conventional solvents, according to their hydrophobic/hydrophilic nature. At the bottom of the scale (lowest hydrophobicity) is the aqueous salt solution. Acetone comes above most aqueous two-phase systems, with hydrophobicity increasing up to heptane. It is possible to have an extremely selective separation of substances using aqueous polymer systems. Aqueous two-phase systems provide a gentle and protective environment for biological material, since both phases are composed primarily of water (Abott et al., 1990; Baskir et al., 1987; 1989).

Polyethylene glycol is one of the most useful polymers in ATPSs. Its solubilization in water is attributed to the attachment of water molecules to many or all of the ether oxygen sites along the polyethylene oxide chain. This attachment occurs by a hydrogen-bonding mechanism. It was found that the addition of monovalent cations to polyethylene-oxide products decreases their solubility; this decrease in the cloud point happens when the competition of salt ions for water effectively reduces the amount of free water available to solubilize the polyethylene. Some inorganic salts are more able to promote this effect (e.g., CaCl₂, MgCl₂, AlCl₃) when the ions form association complexes with the ether groups.

According to Cleland et al. (1992), polyethylene glycol can significantly enhance the refolding of recombinant proteins when accumulated in the form of inclusion bodies that need to be solubilized and refolded to recover activity.

Amongst the variety of aqueous two-phase systems, PEG-dextran is the best-studied system for liquid-liquid partitioning of proteins, in contrast to PEG-salt systems. The effects of polymer molecular mass, concentration, phase density and the presence of salts in aqueous two- phase systems have been studied by many authors: Albertsson (1971; 1986), Johansson (1985a; 1985b), Brooks et al. (1985), Bamberger et al. (1984), Zaslavsky et al. (1978; 1982a; 1982b; 1983; 1992a; 1992b), Carlsson (1988), Huddleston et al. (1991a; 1991b), Asenjo et al. (1991) and Franco et al. (1996a).

Separation of other molecules, including proteins in aqueous two-phase systems, is generally considered to depend on the molecular surface characteristics of the compounds to be partitioned, such as charge, size and hydrophobic properties (Albertsson, 1971; LaMarca et al., 1990). Table 1 presents a list of some biomolecules purified in the last ten years.

Extraction by an ATPS offers advantages for processing on a large scale, such as the possibility of obtaining a high yield, the possibility of continuous processing (Flygare et al., 1990; Enfors et al., 1992; Rostami-Jafarabad et al., 1992a,1992b; Coimbra et al., 1994; 1998; Tomaska et al., 1995; Pawar et al., 1997; Porto et al., 1997) and a reduction in operational cost in relation to the costs of conventional processes (Kula, 1990).

PEG-DEXTRAN SYSTEMS

Effect of Polymer Molecular Mass (MM)

An increase in the molecular mass of dextran or of PEG will lower the concentration required for phase separation. The polymer molecular mass influences protein partitioning as a direct result of interactions between the two polymers. It has been found that the partitioned protein behaves as if it were more attracted by smaller polymer sizes and more repelled by larger polymers, provided all other factors such as polymer concentrations, salt composition, temperature and pH are kept constant. It was observed that smaller protein molecules and amino acids were not affected as much as larger ones. For some proteins (Albertsson et al., 1987) the partition coefficients increased as the MM of dextran increased if all other conditions were kept constant, but little effect was found for low MM proteins (Cytochrome C, 16,000). When the same proteins were partitioned in systems with different PEG MM, their partition coefficients decreased as the PEG MM increased, and for cytochrome C the effect was the smallest. This was attributed to the fact that when the PEG MM is increased, a weaker repulsion energy is required to cause phase separation. Repulsive interactions between the polymer and the protein become stronger as the polymer MM is increased, resulting in a distribution of the protein towards the phase containing the polymer with an unchanged MM. A weak net repulsion between the proteins and the polymer is sufficient to change the distribution when the polymer MM is changed.

Effect of Polymer Concentration

An increase in polymer concentration seems to increase the density of the bottom dextran-rich phase. This difference in density is linearly correlated to tie-line length (Bamberger et al., 1984).

Diamond and Hsu (1989a; 1989b) found that proteins with MM less than 20,000 showed a linear relationship between the ln K in PEG-dextran systems and a difference in PEG concentration between the phases, for any particular system. They found that it was possible to predict the partitioning of a protein at any concentration in that particular system if one partition coefficient in the system were known.

Niven et al. (1990), however, found that for some proteins the partition coefficient was inversely correlated to phase concentration in a PEG-dextran system, showing that better separation could be achieved at high polymer concentrations. This, however, may also affect the concentration of proteins that can be manipulated in the system as polymer concentration has a directly inverse effect on protein solubility.

Guan et al. (1992) showed that the effect of increasing the tie-line length of a PEG-phosphate system, upon the partitioning of penicillin acylase, is considerably higher at low phosphate concentrations (a higher water content), becoming smaller at high concentrations of phosphate salts. Gradual increases in the tie-line lengths lead to enhanced partition coefficients.

Effect of Salts

Salts can affect protein partitioning in different ways in PEG-dextran systems: one is by altering the physical properties of the systems (tie-line lengths) (Kula, 1979, 1985; Chen, 1992) the hydrophobic difference between the phases (Zaslavsky et al., 1982b) and the other is by the partitioning of ions between the phases, which affects the partitioning of proteins according to their molecular charge (Albertsson, 1971; Walter et al., 1972).

Salts have been added to PEG-dextran systems to increase the selectivity of protein partitioning in the aqueous two-phase methodology application for biological separations (Johansson, 1970, 1973, 1974, 1985a; Albertsson, 1971; Hustedt et al., 1978, 1985; Schmidt et al., 1994; Franco et al., 1996a).

(a) Difference in Salt Partitioning and Electrica Potential Between the Two Phases

It was observed that salt ions partition differently between the phases, causing an uneven distribution in the system (Albertsson, 1971; Johansson, 1970, 1974, 1985a, 1985b; Sasakawa and Walter, 1972; Reitherman et al., 1973) that generates a difference in electrical potential between the phases. This difference in electrical potential would be independent of salt concentration, but linearly dependent on the partition behaviour of the ions.

It was also observed by Johansson (1974) that polyvalent anions such as phosphate, sulphate and citrate partitioned preferentially into dextran-rich phases, while halides partitioned nearly equally. Albertsson (1971) showed that for various inorganic salts the partitioning of all negatively charged materials followed the same order as the Hofmeister series. Walter et al. (1972) demonstrated that this effect was reversed when positively charged materials were partitioned. As an example, negatively charged materials have higher partition coefficients in phases containing sodium sulphate rather than sodium chloride, while the reverse holds for positively charged materials. Partition coefficients of negatively charged materials decrease when the cationic series is changed from lithium to sodium to potassium (Johansson, 1974). The ratio between the phosphate ions, rather than the concentration, was decisive for the difference in electrical potential. This applies to multivalent ions, which show a series of pH-dependent dissociations and was clearly the reason for the potential difference found between the two phases (Kula et al., 1982).

(b) Effect of Salts on the Hydrophobicity of the ATPS

Kula et al. (1982) observed that in the presence of PEG and dextran phosphate ions and sulphate ions accelerated a gel formation, which was pH dependent. It was attributed to a complex formation of spaced hydroxyl groups in the polyglucan backbone of dextran. This association of dextran chains increased the exclusion volume, which in turn might increase the partition coefficient of any protein of a given size. Lee and Sandler (1990) observed that the partition coefficient of the antibiotic vancomycin (MM 1,448, pI 8.1) in PEG-dextran systems at pH 7.0 increased exponentially with either NaCl or sodium sulphate concentrations, rather than decrease with sodium sulphate, as would be expected if electrostatic effects were dominant. This effect was attributed to possible hydrophobic interactions between vancomycin and PEG and electrostatic interactions.

Zaslavsky et al. (1982a) found that the partition coefficient of ions was dependent on their own concentration, in the range of 0 to 285 mM in PEG-dextran systems, and also on the type of ion present. This followed the order NaH₂PO₄á sodium phosphate pH 6.8 á Na₂HPO₄. An electrical potential of 3.0 mV, created by 0.01 M of HPO₄^--H₂PO₄⁼, was almost neutralized by a NaCl concentration of 0.15 M. Their conclusion was that in a PEG-dextran system containing 0.01 M sodium phosphate buffer pH 6.8, small amounts of NaCl reduced the difference between the relative hydrophobicity of the phases and this difference remained the same up to 0.12 moles/kg. A further increase in NaCl concentration to 0.15 increases this difference, which appears to be invariable in the range of 0.15 to 0.5 moles/kg. A slight increase in relative difference occurs at higher concentrations at higher salt concentrations (e.g., 1.0 M). The phase hydrophobicity measurements were carried out by calculating the free energy of transfer of CH₂ groups from one phase to the other and from one phase to a reference solution (Zaslavsky et al., 1982a, 1982., 1983).

Bamberger et al. (1984) and Brooks et al. (1984) observed that some ions (e.g., SO₄⁼ and PO₄⁼) had a greater tendency to leave the PEG phase than others (F^-, Cl^-, Br ^-, I^-, ClO^-) and that some others correlations (difference in electrical potential between the phases and tie-line lengths) were direct functions of these effects. NO₃^- and Cl^- did not leave any phase.

(c) Combined Effects of Salts upon Protein Partitioning in PEG-Dextran Systems

Most of the problems with testing the predicted charge dependence of K are difficult to recognize due to the accompanying changes in phase composition, such as variation in pH and salt concentration.

Depending on the distribution of charged ions, the phases will be attractive or repulsive to proteins (poly-ions) so the proteins will tend to move towards the phases with the opposite charge. The electrostatic interactions (attraction/repulsion) between the phases and the proteins will only be detected if other important factors, such as hydrophobicity, bioaffinity, etc., do not overwhelm them. The electrostatic effects in aqueous two-phase systems for protein partitioning can be observed at low salt concentrations up to 0.10 mol/kg of the phase. This is like the "salting in" effect, which typically involves low salt concentrations and is based on electrostatic interactions (Melander and Horvath, 1977; Scopes, 1994).

The difference in electrical potential is a rather controversial subject (Zaslavsky et al., 1982b). According to these authors, the electrical potential represents the difference in hydration of the ions taking part in the distribution equilibrium, and hence, seems to represent (in a rather limited way) the difference in the relative hydration abilities of the two phases of the system. According to Brooks et al. (1984), absolute potential measurements cannot be made in a strict thermodynamic sense due to the unknown effect of the liquid junction potentials at the tips of the salt bridges in the electrode measuring it. According to Brooks et al. (1984) and Zaslavsky et al. (1982b), it is therefore necessary to demonstrate that the measured potentials are consistent with the partition coefficients observed in the ATPS. Pfennig et al. (1998) have observed that the electrostatic potential difference between coexisting phases is a common property at interfaces even though the phases are strictly electroneutral and can not be measured, however it can be quantified under controlled conditions.

PEG-SALT SYSTEMS

The formation of PEG-salt systems was first observed by Albertsson in the 1950s, but the theoretical fundamentals have not been well explained. Boucher and Hines (1976) found that for PEG solutions the addition of some inorganic salts (sulphates and carbonates) is more effective than the addition of others in reducing the critical concentration of cloud point curves. Ananthapadmanbhan and Goddard (1987) found that inorganic salts dramatically reduced the PEG cloud point at high temperatures.

PEG-salts systems have been introduced for the practical application of large-scale protein separation because of the larger droplet size, greater difference in density between the phases, lower viscosity and lower costs, leading to a much faster separation than in PEG-dextran systems. Industrial application of PEG-salt systems was improved by the availability of commercial separators, which allowed faster continuous protein separations (Kroner and Kula, 1978; Kroner et al., 1982; Kula et al., 1982; Kula, 1990).

For PEG-salt systems, salting-out effects appear to operate with increasing tie-line length, shifting proteins from the salt phase into the PEG-rich phase, or if protein solubility in the PEG phase is not sufficient, they tend to precipitate at the interface. Solubility and salting-out limits are dependent on the properties of individual proteins; therefore, a differential response is expected when a mixture of proteins is handled (Kula et al., 1982; Andrews and Asenjo, 1989).

Köhler et al. (1991) studied precipitation curves for ß-galactosidase and four others samples of ß-galactosidase fused to small proteins (SpAßgal) in PEG and in phosphate solution and found they were strongly related to protein partitioning in PEG-phosphate two-phase systems. It was suggested that the considerable increases in the partition coefficient of SpAßgal at longer tie-line lengths were due to the loss of solubility in the phosphate phase followed by adsorption at the interface, rather than an increase in the partition coefficient as such.

Initially PEG-phosphate systems were widely used (Kula, 1979, 1985, 1990; Kula et al., 1982; Hustedt and Kula, 1977; Hustedt et al., 1978; Veide et al., 1983). Greve and Kula (1991) have studied ways of recycling the phosphate phase of the systems to minimize environmental pollution. The recycling of the phosphate phase was achieved by its separation from the solids by the use of alcohols. PEG from the top PEG-rich phase can also be successfully recycled (Kroner and Kula, 1978; Kroner et al., 1982; Hustedt et al., 1985, 1988; Hustedt, 1986).

More recently PEG-sulphate systems have begun to be used. Chiang and Wang (1988) used them for recuperation of L-aspartase from fumarase, produced by E.coli. The best separation was achieved with PEG 4000 and (NH₄)₂SO₄ at pH 7-7.5. The presence of 2% NaCl (0.17 M) made the separation much worse. With 4% NaCl (0.34 M), a poor separation was obtained (a tenfold decrease in K for aspartase). Since a pH or phase ratio change was not observed, the dramatic change in K was considered to be due to a change in hydrophobicity between the phases. Lee and Sandler (1990) found that the partitioning of antibiotic vancomycin in PEG 8000-phosphate systems was exponentially increased by addition of NaCl (0 to 1.5 moles/kg) or sodium sulphate (0 to 0.7 moles/kg), but not significantly changed by thiocyanate. It was suggested that the increased partitioning of vancomycin was due to hydrophobic interactions promoted by water-structure-making salts in combination with potassium phosphate (from the phase components), which would reduce the water activity and alter the phase composition.

Eiteman and Gainer (1990) studied the influence of the difference in composition between the phases on the partitioning of small alcohols and peptides. A model for protein partitioning was suggested, taking into account the difference in PEG concentration between the phases and the solute hydrophobicity relative to the hydrophobic difference between the two phases.

Citrate systems were developed in order to make ATPSs less harmful to the environment. Citrate is biodegradable and non-toxic, and it could be discharged into a wastewater treatment plant (Vernau and Kula, 1990).

MODELS FOR PARTITIONING BETWEEN THE PHASES

Bronsted Theory

The basis for separation by a two-phase system is the selective distribution of substances between the phases. For soluble substances, distribution takes place mainly between the two bulk phases. Distribution is characterized by the partition coefficient, K, defined as the concentration in the top lighter phase, C_top, divided by the concentration in the heavier bottom phase, C_bottom:

K= C_top /C_bottom (1)

Obviously the choice of a suitable phase system is the key step in all partitioning work.

If the energy needed to move a protein molecule from one phase to another is DE, one would expect that at equilibrium a relationship between the partition coefficient and DE would be expressed as follows (Baskir et al., 1987):

C₁/C₂ = e ^{-DE/k T} (2)

where k is the Boltzman constant and T the absolute temperature, and C₁ and C₂ are the concentration of protein molecules in phases 1 and 2.

DE must depend on the size of the particle or molecule, since the larger the size, the greater the number of exposed atoms which can interact with the surrounding phase, and the following formula was suggested:

C₁/C₂ = e ^lM/k.T (3)

where M is MM and l in this case is a factor that depends on properties other than MM. For a spherical molecule, M could be replaced by A, the surface area of the molecule:

C₁/C₂ = e ^l A/k.T (4)

and l in this case is a factor that depends on properties other than surface area, for example surface properties as expressed by the surface free energy per unit area.

This is known as the Bronsted partition theory, and its main point is the exponential relationship between the partition coefficient and properties that enter into the l factor, for example hydrophobicity or affinity and charge. Small changes in such factors will cause relatively large changes in the partition coefficient. The theory therefore predicts a high degree of selectivity. Although the Bronsted model demonstrated why protein partitioning may be sensitive to particle MM, it is at best a qualitative model, since it combines all the other system variables into a single parameter.

Albertsson’s Model

Albertsson (1971) derived an expression for the dependence of the distribution potential difference, y , between the two phases on the buffering salt:

(5)

R represents the gas constant

T represents absolute temperature

K⁺ represents the cation salt partition

K^- represents the anion salt partition

Z⁺ and Z^- represent the number of charges on the salt ions (e.g., valence)

y represents the electrical distribution potential

This is also a qualitative model, and it demonstrates the high exponential dependence of protein partitioning on its charge, surface area, surface energy and difference in distribution potential between the phases.

In practice, however, there has been no consistent and generalized effort to use these theories to predict partitioning behaviour, so they have been used only as descriptive tools to explain observed phenomena.

These models are limited to examining only the effect of protein molecule characteristics without attempting to analyze in detail the contribution of phase environment. These theories also give no indication of how molecular mass, concentration or the choice of phase polymer will influence partitioning in aqueous two-phase systems.

Lattice Model Proposed by Brooks et al. (1985) (Model for PEG-Dextran Systems)

The increased interest in ATPSs for a wide range of applications, combined with the lack of a useful predictive model for protein partitioning, has led to an interest in the development of models. Brooks et al. (1985) developed a lattice model by extending the theory of polymer-polymer solvent mixing to multicomponent systems. In this case, the system is considered to be a four-component system, containing water (component 1) and three polymeric solutes (components 2, 3, and 4), one of which is biomaterial (component 4). It is assumed that there is a very low concentration of biomaterial relative to the other components, and all the polymeric components are assumed to be equally soluble in the solvent. The Brooks model qualitatively demonstrates several of the trends found empirically. Less partitioning occurs between the phases, even for large biomaterials. Also the value of K is higher for greater differences in polymer concentration between the phases. All other factors being equal, the protein will partition in favour of the phase containing the polymer with a lower MM. Their model provides some basis for understanding biomaterial partitioning, but Brooks et al. pointed out that the physical picture of the biomaterial as a random coiling polymer is unrealistic. It is well known that like most biological molecules, proteins are generally tightly folded, compact globular structures which contain a large proportion of the polypeptide shielded internally from contact with the surrounding solution. One should expect that real native proteins would have much lower surface contact areas than random coiling polymers. In addition, the nature of steric interactions between the phases and the biomaterial will be different if a compact hard body model, rather than a random coil, is assumed.

The Virial Expansion Model

King et al. (1988) developed a model for polymer-polymer systems that assumes a general form for the chemical potential of the components, and various terms are determined by independent measurement, allowing the calculation of the phase diagram and the protein partitioning coefficient with it. The model states that protein partitioning is a function of polymer concentration and the interactions between the polymers and the protein. The virial expansion model can be modified and used to predict the effect of moderate concentrations of salts on protein partitioning. It gives a qualitative relationship between K and polymer concentrations and information about the volume "excluded" by each component in solution, which is dependent on the molecular mass of the component. Measurement of the interaction coefficient by low-angle light scattering indicates that protein-polymer interactions are generally only slightly affected by the presence of different salts. The protein-protein interaction coefficient for many proteins has been found to be dependent upon the choice of salt. The dependence of protein-protein interactions on salt type could be due to electrostatic interactions, stabilization of the protein, and the interaction of the salt solution with the hydrophobic portions of the protein (Melander and Horvath, 1977). This model, however, is not fully understood and is only applicable to polymer-polymer systems and not to polymer-salt systems.

Haynes et al. (1989a; 1989b) improved King’s model, developing a molecular thermodynamic model to predict properties of aqueous two-phase systems containing polymers, electrolytes and proteins. According to the Debye-Hückel theory, at concentrations of electrolyte in solution of less than 0.1 M, electrostatic interactions can be well explained. Haynes' model uses this theory and independent measurements of coefficients of polymer-polymer, ion-ion and ion-polymer interactions. The model can predict salt ion partitioning, which is inversely correlated with tie-line length (TLL) and ion size and is also in agreement with the Hofmeister series. Electrical potential differences were predicted as a function of TLL, and binodial curves of phase diagrams were in good agreement with experimental data. The shortcomings of their model were the unreal ratio between the size of protein-ion interactions, which are in disagreement with the Debye-Hückel theory, and the uneven distribution of charge over the protein surface. It also had to be improved for higher polymer concentrations.

Cabezas et al. (1989; 1990; 1991) developed a statistical mechanism model for the prediction of phase diagrams of aqueous two-phase systems. It incorporated the dependence of the phase diagram on polymer molecular mass and polydispersity. The model was in agreement with independent thermodynamic calculations from experimental data.

Model for Polymer-Salt Systems

A model for polymer-salt systems was developed, indicating that the polymer concentration of the salt-rich phase of the ATPS can be neglected, since it is generally small compared to the salt concentration. The protein solubility term is expressed by a relationship between protein solubility in the salt-rich phase, the molar salt concentration in the salt-rich phase, the solubility of the protein in pure water and the salting-out constant of the salt for the protein. The salting-out constant depends on the protein and the salt and is related to the lyotropic series (Baskir et al., 1987, 1989). In the polymer-rich phase, however, the concentration of both polymer and salt are significant. Randomly coiled polymer chains are known to contain a large amount of solvent. The polymer in the polymer-rich phase "excludes" the protein and the salt from a portion of the water present in the phase. The formation of hydrated PEG removes some of the water, which would otherwise be available for dissolving the salt or the protein. Consequently the protein and the salt are concentrated in the portion of available water in the phase.

Eiteman and Gainer Model

A mathematical model was developed by Eiteman and Gainer (1990, 1991) to predict partition coefficients for solutes in PEG-sulphate systems. Their expression has been validated for peptides, showing that increasing the difference in concentration of PEG (Dw) between the phases or increasing solute hydrophobicity also increases the partition coefficient.

log K_hydrophob = D D w log (P/P₀)

An increase in Dw can be considered to be equivalent to an increase in tie-line length. The model still has to be improved for proteins. K values for papain and trypsin increased as Dw increased. K for lysozyme first decreased, and after reaching a minimum, then increased as Dw increased (Franco et al., 1996b).

AFFINITY PARTITIONING

In the last 30 years, several groups have studied methods to increase partitioning by the use of biospecific interactions in ATPSs (Flanagan and Barondes, 1975; Johansson et al., 1983, 1984; Luong and Nguyen, 1990).

The initial works on affinity partitioning in ATPSs were to purify trypsin by using PEG-bound ligand p-aminobenzamidine (Takerkar et al., 1974) and S-23 myeloma protein by using dinitrophenol as ligand (Flanagan and Barondes, 1975).

The degree of affinity partitioning, K_aff, can be described by the ratio between the partition coefficients of proteins with and without a ligand (Johansson, 1985b):

This equation describes the increase in the partition coefficient of a protein by the binding of a specific ligand to the PEG-rich phase.

Affinity partitioning results in specific extractions of proteins, nucleic acids, membranes, organelles and even cells, mainly when biospecific ligands are used (Walter et al., 1985).

The ligand can be a natural substance or a synthesized molecule. There are ligands that interact selectively with proteins due to the specific sites. There are many groups of ligands, such as hydrophobic and thiophilic ligands (Porath et al., 1975), metal chelates (Wuenschell et al., 1990; Arnold, 1991; Birkenmeier et al., 1991; Zutautas et al., 1992; Chung et al., 1994), quaternary ammonium compounds (Johansson et al., 1981), dyes (Kopperschläger and Johansson, 1982; Kroner et al., 1982; Johansson et al., 1983; Johansson and Andersson, 1984a; Johansson and Andersson, 1984b; Johansson et al., 1984; Birkenmeier et al., 1984; Johansson and Joelsson, 1985a, 1985b; Cordes et al., 1987; Giuliano, 1991; Wang et al., 1992; Zutautas et al., 1992; Birkenmeier, 1994; Kopperschläger, 1994; Lin et al., 1996), fatty acids (Johansson and Shambhag, 1984; Johansson et al., 1985), inhibitors (Silva et al., 1997) and monoclonal antibodies (Elling et al., 1990, 1991; Elling and Kula, 1991; Andrews et al., 1996). Natural ligands are generally expensive due to the high cost of purification, chemical and biological lability (Kopperschläger, 1994).

Table 2 reports the purification of different biomolecules by affinity partitioning described in the literature.

Downstream processing of proteins has been considerably advanced with the use of affinity separation methods, as can be evidenced by the extensive publications in this field.

Application of affinity separation methods in the initial steps of the extraction process was initially described by Sada (1990). The concept of the use of affinity interactions for initial isolation is attractive as a technique of high resolution applied early in the purification process, reducing the volume of material to be manipulated later on, the consumption of chemical products used in the process and biological activity loss of biomolecule (Asenjo and Patrick, 1995).

According to Kopperschläger (1994), this technique can operate continuously and be scaled up. Also, the target biomolecule can be purified by a single or multistep extraction, and affinity partitioning of biomolecules is a simple and adequate approach to the qualitative and quantitative study of protein-ligand interaction.

Blennow (1994) showed the following advantages of using affinity partitioning in ATPSs: 1) enzyme recovery is high, 2) the conditions are mild, 3) the procedure can be scaled up, 4) the method is reasonably inexpensive and 5) standard laboratory devices can be used.

Arnold (1991) reports the following advantages of metal-affinity partitioning in ATPSs over affinity chromatography for protein purification: 1) metals can be recycled a large number of times, 2) high metal loadings and high protein capacities then can be attained, 3) product elution is achieved relatively easily, 4) ligands are regenerated and 5) metal chelate ligands are cheap.

CONTINUOUS EXTRACTION IN ATPSs AND RECYCLING OF PHASE COMPONENTS

Recent advances in ATPSs have enhanced the choices of commercial applicability for large-scale operation (Winter et al., 1999; Lorch, 1999). However, as the main properties of ATPSs are quite extreme when compared with non-polar solvents (Table 3), detailed studies had to be conducted in order to adapt the extraction equipment to aqueous two-phase systems. Very fine droplets are formed in a continuous extraction column, allowing a very high interfacial area for the rapid transfer of enzyme or protein (Bhawsar et al., 1994).

Enzyme mass transfer was investigated in a sieve-plate extraction column (Bhawsar et al., 1994), in a packed extraction column (Patil et al., 1991), in a modified spray column (Pawar et al., 1997) and in a conventional spray column (Sawant et al., 1990).

The effect of design parameters (number of plates or plate spacing) and operating parameters (dispersed phase velocity and physical properties) on the overall volumetric mass transfer coefficient and hold-up have been studied by some authors (Sawant et al., 1990; Pawar et al., 1997; Bhawsar et al., 1994; Patil et al., 1991). It was observed that the viscosity of the dispersed phase was usually more than 14 mPa s, which is much higher than that in conventional liquid-liquid extraction. Also, the difference in density of a non-polar system is at least twice that in the case of ATPSs. In addition, the diffusibility of enzymes and proteins in ATPSs is about one order of magnitude lower than that in conventional systems. Bhawsar et al. (1994) studied the enzyme mass transfer in a sieve-plate extraction column and observed that the total hold-up, the fractional dispersed hold-up and the volumetric mass transfer coefficient increased as the orifice diameter and the number of orifices on plates in the sieve-plate extraction column increased. The values of the mass transfer coefficient and total hold-up also increased as the number of plates in the column increased together with a simultaneous decrease in the plate spacing. It was also found that these three parameters decreased as the tie-line length (higher concentrations) of the ATPSs increased and that the PEG-rich dispersed phase controlled the resistance to mass transfer. It was observed that the major contributions to mass transfer of amyloglucosidase were attributed to the drop rise and the coalescent stages. The drop formation contribution was found to be negligeble.

The recovery of polymers after extraction of a biomolecule is a procedure as important which is in laboratorial processes as it is on an industrial scale. The ability to easily recycle the polymers would make large-scale ATPSs more economically attractive (Carlsson, 1988).

Methods of recycling the phase-forming components in ATPSs have been used on small scale, as described by different authors (Carlsson, 1988; Kula et al., 1982). Carlsson (1988) reports that PEG is recycled by extraction with chloroform and reused in a new phase system. Another alternative mentioned by this author is the transfer of a PEG-rich phase of a system where the biomolecules had partitioned in a first system to a second fresh saline phase with a different composition. The biomolecules would then partition into the bottom-rich phase of this second system, releasing a protein-free PEG-rich phase that would be reused in another extraction process.

Kula et al. (1982) described some methods to recycle PEG, such as ultrafiltration or dialysis. The other way to obtain PEG-free enzymes is the adsorption of the enzyme by suitable adsorbents, thus recovering the PEG phase.

Greve and Kula (1991) studied three ways to recycle salt and the best was by using an aqueous ethanol solution to extract salt. The alcohol and salt-rich phase flow to an evaporator where the alcohol is removed and recycled. The salt solution is concentrated, if necessary, in a second evaporator, from which most of the water may also be recycled. The concentrated salt solution is then reintroduced into the protein extraction process.

Recently van Berlo et al. (1998) have demonstrated that ammonium carbamate in combination with polyethylene glycol (PEG) produces ATPS at 25⁰C and atmospheric pressure. PEG with molecular masses of 2,000 and 4,000 are suitable phase-forming polymers, but PEG 10,000 is less suitable due to the high viscosity of the top phase and the very small difference in density between the two phases. Ammonium carbamate is a volatile salt that can be recycled and sent to the extraction system as gaseous carbon dioxide and ammonia.

CONCLUSION

An understanding of the main ATPS fundamentals has led to broader applications of this type of system in the extraction and separation of biomolecules. The chronology of ATPS can be roughly divided into three main stages. Initially the partitioning of small ions of different salts were observed in polymer-polymer systems. The effect of the uneven partitioning of ions upon the partitioning of charged proteins was noticed, and empirical models were developed to explain this behavior. Larger-scale applications were found to be feasible for the extraction of intracellular and extracellular products. Some equipment already employed in conventional liquid-liquid extraction has recently been adapted to ATPS processes, but more practical equipment is still being studied. Also, ATPS have been successfully developed for specific needs mainly affinity extraction of priced biomolecules. The description and understanding of the mass transfer of particulate material, such as macromolecules in polymer-salt continuous systems, still need to be improved. Industrial application of the extraction of target molecules from cell debris and inclusion bodies needs to be further developed.

ACKNOWLEDGMENTS

Financial support from FAPESP and CNPq is gratefully acknowledged.

ABREVIATIONS

K Partition coefficient

PEG Polyethylene glycol

R Gas constant

T Absolute temperature

y Electrical distribution potential

Dw Difference in concentration of PEG

MM Molecular mass

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*

To whom correspondece should be addressed

Publication Dates

• Publication in this collection
  24 Apr 2000
• Date of issue
  Mar 2000

History

• Accepted
  27 July 1999
• Received
  24 May 1999