ANALYTICAL
BIOCHEMISTRY
197,
l-18
(19%)
REVIEW Partitioning in Aqueous Two-Phase Systems: Recent Results Harry Walter,* G&e Johansson,? and Donald E. Brooks$ *Laboratory of Chemical Biology, Veterans Affairs Medical Center, Long Beach, California 90822; TDepartment Chemical Center, University of Lund, S-221 00 Lund, Sweden; and SDepartments of Pathology and Chemistry, University of British Columbia, Vancouver V6T 1 W5, British Columbia, Canada
From analytical to commercial scale, aqueous twophase systems have a niche in the purification, characterization, and study of biomaterials (1). Initially described by Beijerinck toward the end of last century, such phase systems were rediscovered and first employed in the 1950s by Albertsson for partitioning biomaterials (2,3). Aqueous two-phase systems are generally composed of a water solution of two structurally distinct hydrophilic polymers or of one polymer and certain salts (e.g., alkali phosphates). Above critical concentrations of these components, spontaneous phase separation takes place with each of the two resulting phases enriched with respect to one of the components. The replacement of organic by aqueous solutions in immiscible two-phase systems has permitted the application of the classical separatory method of resolving components of a mixture by partitioning not only to labile macromolecules but also to cells, membranes, and organelles. Two-polymer aqueous phase systems can be buffered and, if necessary, rendered isotonic. Appropriate selection of polymers [the most widely used being dextran (Dx)’ and polyethylene glycol (PEG)] results in
i Abbreviations used: Ab, antibody; CCD, countercurrent distribution; Cross-point (isoelectric point), obtained by cross-partitioning, i.e., the pH at which two plots of Kvs pH for a material intersect when the K’s are measured in phase systems containing different salts but the same polymer concentrations; DEAE-, diethylaminoethyl-; DMSO, dimethylsulfoxide; Dx, dextran; EHEC, ethylhydroxyethylcellulose; FACS, fluorescence-activated cell sorter; FA-PEG, polyethylene glycol fatty acid esters; Fi, Ficoll; G, apparent partition coefficient (or ratio), obtained from the location of the peak in a CCD curve = r-l(n - rma.), where r,, is the tube (or cavity) number of the peak of the distribution and n is the number of transfers; HPD, hydroxypropyldextran; HPS, hydroxypropylstarch; IDA, iminodiacetic acid; IO, inside out; K, partition coefficient (used with materials that partition between the two bulk phases) = concentration of material in top phase/concentration of material in bottom phase; P, partition ratio (used with materials, e.g., cells, which partition between a bulk phase 0003-2697191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
of Biochemistry,
phase systems which are mild and nondeleterious to most biomaterials partitioned in them. These phases have interfacial tensions several orders of magnitude lower than those of aqueous-organic or organic-organic systems. The manipulation of polymer and ion composition and concentration determines the physical properties of the phases which, in turn, effect separations of biomaterials based on different physical parameters. For example, even though Dx and PEG are themselves nonionic, certain salts (e.g., phosphates, sulfates) have different affinities for the two phases, a phenomenon which gives rise to a Donnan potential between the phases and causes them to be charge-sensitive. Other salts have equal affinities for the two phases and give rise to non-charge-sensitive systems. The incorporation of a ligand bound to one of the phase-forming polymers (or one which partitions or is made to partition extremely into one of the phases) permits affinity partitioning to be carried out. Partitioning of biological particulates has proved to be an extremely sensitive method for their separation and fractionation. The partitioning of larger particulates depends predominantly on their surface properties. By carrying out multiple extractions [e.g., countercurrent distribution (CCD)] even subtle surface alterations of cells that accompany normal and abnormal in uiuo processes (e.g., differentiation, maturation, aging, metastatic potential) or in vitro treatments can be traced as these are often reflected in altered partition ratios, i.e., P values (l-3). Membranes and organelles can be fractionated, plasma membranes can be purified, and membrane domains charted. Right-side out and inside out vesicles can be separated. Proteins, including isoenzymes, have been efficiently segregated while afand the interface) = quantity of material in bulk phase as a percentage of total material added, PS, photosystem; PVA, polyvinyl alcohol; PVP, polyvinylpyrrolidone; RO, right-side out; TMA-, trimethylamino. 1
Inc. reserved.
2
WALTER,
JOHANSSON,
finity partitioning has permitted the specific extraction of a multitude of proteins including dehydrogenases and kinases and the fractionation of nucleic acids on the basis of base- and sequence-specificity (l-4). The aqueous phases obtained with one polymer, usually PEG, and certain salts, which are not isotonic, have found their main use in the rapidly expanding biotechnology of protein purification (4). Pa~icularly advantageous is the fact that extractions can easily be scaled up. Because the high salt concentration of such systems often precludes affinity partitioning and because of the prohibitively high cost of dextran for commercial applications, other polymers (i.e., Dx substitutes) or polymer combinations are being examined for use in two-polymer aqueous phases. Other biotechnological applications of aqueous two-phase systems include extractive bioconversion processes and the concentration of biomaterials such as viruses. Since the parameters that determine the partition coefficient, I( (or the partition ratio, P), of materials are exponentially related to the K (or P) value, partitioning often yields separations and information on physical properties of biomaterials not readily obtained by other means. Here we relate some of the rapid advances in and novel applications of aqueous phase pa~itioning during the past 5 years, i.e., since the previous review (l), as well as theoretical aspects of phase separation and partitioning. It is gratifying that some of the “prospects” we noted (1) have come to pass. These include: finding inexpensive polymers with favorable properties, development of biospecific extraction methods (also for cells), new apparatuses for more rapid processing of materials, use of highly efficient columns for protein (and nucleic acid) fractionation based on two-polymer aqueous phases, development of systems for use at high and low temperatures, addition of small quantities of water-soluble organic solvents to aqueous phase systems to permit partitioning of materials with low solubility in water, and use of systems composed of more than two phases. THEORY
OF PETITIONING
PHENOMENA
Considerable advances in the theory of two-phase systems and partitioning phenomena have been made since this subject was last treated (5). These advances derive largely from the impetus provided by the potential of partitioning in biotechnology, particularly as one or more steps in the medium to large scale isolations of genetically engineered proteins. The chemical engineering community has been dealing with liquid-liquid extraction problems for decades (6) and has found in aqueous phase partitioning a fertile area for investigation. Experience in the industrial application of extraction has shown clearly that deeper understanding leads
AND
BROOKS
to more efficient separations and more profitable processes. The goal of many of the groups involved is to secure a sufficient understanding of macromolecular partitioning to allow prediction of K for a defined material under arbitrary conditions and hence to allow optimization of separation. In some of the approaches currently being pursued achievement of this goal implies an ability to predict phase behavior of the systems, i.e., to predict phase diagrams, on the basis of a minimum number of experimental measurements of characteristic constants describing interactions between pairs of elements in the system (7-10). Other groups have assumed the presence of an invariant two-phase system and concentrated on modeling the interactions between phase components which result in the observed K (11-14). In all cases to be reviewed significant agreement with experiment exists under some sets of conditions. However, no single approach has proven so superior that all would accept its universality. Due to space li~tations we will provide only a broad overview of the theoretical developments since 1985. For a more detailed discussion the excellent recent review by Abbott et al. (15) is recommended. Prediction of Phase
Diagrams
A sufficiently detailed understanding of the interactions which cause phase separation would obviously contribute greatly to our capacity to predict the K of added material, particularly if the partitioned material was a low molecular weight species or another polymer. Two approaches to the prediction of phase diagrams have been taken, one based on polymer solution theory and the other adapted from the thermodynamic treatments of liquid phase equilibria. In neither case have predictions of phase behavior been made from first principles, i.e., simply from the chemical structures of the components; liquid state theories are not available to describe such complex systems. Rather, various theoretical constructs are applied to sets of experimental data to evaluate the constants necessary to allow prediction of phase diagrams of related systems. Generally, the goal is to maximize predictive power with the minimum number of measured constants. The best known polymer solution treatment is the lattice theory of Flory and Huggins (5) which has been quite successful in predicting general properties of polymer solutions in unstructured solvents. In such models the solution is represented by a three-~mensional lattice, each site of which is filled with a solvent molecule or a segment of one of the polymers present. The free energy of mixing of the solution is evaluated from the sum of the energies of interaction between the various segments and solvents and from the configurational entropy associated with the numbers of ways of arranging the components on the lattice. If in a two-polymer, one-
RECENT
RESULTS
IN
AQUEOUS
solvent mixture the interaction energy between unlike polymer segments is even modestly unfavorable, the total free energy can be lowered-at the cost of an entropy decrease-if the number of such contacts is minimized by the formation of two phases, each rich in one of the polymers (5). In this lattice model the parameters which describe the system are the molar volumes of the polymers relative to the solvent molar volume, proportional to polymer molecular weights, and the interaction constants, xi j, which are proportional to the energy change which occurs on forming from pure components contacts between solvent (component 1) and either type of polymer segment (xl2 or x13) or contacts between the two-polymer segment types (xZ3). The derivation assumes the average polymer segment density is constant throughout the solution. Given the simplicity of the approach it has been remarkably successful at describing the thermodynamic properties of amorphous polymer mixtures and solutions. The main problem with applying this theory to aqueous polymer solution phase separation is that no solvent structuring effects are taken into account. While some ordering of solvent in contact with polymer can be introduced by allowing the interaction energy to contain a local entropy component the result is not well suited to strongly hydrogen bonding solvents like water. In fact, one whole class of phase separation phenomena, known as LCST behavior, cannot be described by the Flory-Huggins theory (16). This occurs when solutions of certain polymers such as PEG [or detergents with PEG headgroups (17,18)] are heated, reducing the Hbonding with water which holds them in solution. Nonetheless, the lattice theory formalism has been applied to fill out Dx-PEG-water phase diagrams using empirical values for the interaction parameters determined from one set of phase compositions and reasonable results were obtained (19). The values of the interaction parameters obtained do not appear to be applicable to other types of thermodynamic predictions, however [compare xij parameters with (ll)], which indicates the FloryHuggins theory is not accurately describing the details of the interactions present. An alternate approach is to apply what is known as the osmotic virial expansion in which thermodynamic functions, in particular the osmotic pressure or solvent chemical potential, are described by a power series in the polymer concentrations with empirically determined coefficients. The constants in the osmotic pressure expression which multiply the second order concentration terms, aij, are known as second virial coefficients and are related to the energies of interaction between the pairs of species represented by i and j. For instance, to describe two polymers in water the virial coefficients az2, us3, and az3 would have to be evaluated if the power series is taken to second order. The degree of compatibility or incompatibility between the
PHASE
3
PARTITIONING
polymers therefore is described by the sign and magnitude of uZ3. The method was first applied to describe phase separation of polymer (and protein) solutions by Edmond and Ogston (20) who found it could satisfactorily treat these systems. Recently, more rigorous calculations along the same lines have been made by groups in Berkeley (9,lO) and Arizona (7,B) as part of longer term efforts to predict protein partition. Both groups have taken the vital step of specifically including the effects of salts in their calculations. The two approaches differ in detail, particularly in the treatment of salt effects and polymer molecular weight. The Berkeley computation does not address the molecular weight issue and measures individual virial coefficients for each polymer fraction used. Cabezas et al. (7,8,21), on the other hand, have applied Renormalization Group ideas to allow calculations for all fractions of a given polymer whose molecular weights are known. Both groups use thermodynamic measurements (low angle light scattering, osmotic pressure, isopiestically determined activities) on two- or three-component solutions to obtain all the necessary constants; no adjustable parameters are utilized. Both have had considerable success at predicting phase diagrams for Dx-PEG (9,21), Dx-methylcellulose (21), and DxPEG-salt (8,lO) mixtures. Their most recent published work also includes predictions for the partition of salts in Dx-PEG systems which reproduce experimental data slightly less well than the phase diagrams. Predictions of PEG-salt phase diagrams are just becoming available (22). One unexpected prediction which should be tested experimentally is the dependence of the Dx-PEG phase diagram on very low concentrations of Na,SO, (8). Cabezas et al. claim that even 0.001% Na,SO, can shift the binodal to much lower Dx concentrations, arguing that experimentally no phase system is salt-free to this level so that true zero salt measurements have not been carried out. The published Berkeley calculations (9,lO) do not address this issue. Another area which deserves more consideration is the theoretical effect of polydispersity of the polymer fractions used, particularly the changes in molecular weight distribution which occur in the two phases relative to the source material. Some relevant theoretical work has appeared but it is difficult to apply it in the above formalisms (23,24). Some of the reported fractionation effects are dramatic (24). Prediction of Protein Partition
Coefficients
The first attempt at writing a molecular-level theory for partitioning of macromolecules in a two-polymer aqueous system again utilized the Flory-Huggins formalism, the partitioned material being treated as a third polymer (5). The result, obtained by equating chemical
4
WALTER,
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potentials of the partitioned species in the two phases and retaining only first order terms in the polymer concentration differences, provided an expression for the partition coefficient, K, in terms of the polymer concentration differences between the phases, the molecular weights of the two polymers and the added macromolecule and xij parameters for the macromolecule interacting with the solvent and each polymer: In K = PJ(cbl - d$)O - xlp) + (4: - &)(1/p,
- x2J
+ (4: - 4:) x (l/P, - x3p)l,
111
where
Pi = (molecular
volume of component i)l (molecular volume of solvent) i= 1, 2, 3, or p refers to solvent, polymer 1, polymer 2 or protein, respectively fraction of solution volume occupied by component i; t or b refer to top or bottom parameters describing the Xij = interaction i-j interaction, described above.
phase
Although it is clear that K describes the distribution of a flexible polymer coil, the predictions of the treatment also described most of the qualitative features observed for protein partition in the absence of electrostatic effects (25). That is, partition becomes more one-sided with increasing protein molecular weight and with increasing difference in concentration between the phases of either polymer. Partition also shifts in favor of a phase in which the polymer molecular weight is reduced, the more so the higher the protein molecular weight. The agreement with trends observed when polymer or protein molecular weights are changed may be fortuitous since the protein segments are neither uniformly distributed nor free to occupy large numbers of configurations, as assumed in the derivation. In the limit of low molecular weight proteins, i.e., for oligopeptides, the theory is much more realistic. A simplified version of the Flory-Huggins expression has been derived (26), utilizing the observation that in most Dx-PEG systems [but not in all two polymer mixtures (2)] the difference in Dx concentration between the phases is proportional to the difference in PEG concentrations. This reflects the fact that the tie lines in these systems are parallel in their phase diagrams. The equation is In K = A*(wi
- WE),
PI
where A* is an empirical parameter which depends on the molecular weights of the polymers and protein and w2 is the weight fraction of PEG in the indicated phase. Equation [2] was found to describe the partition of dipeptides and some low molecular weight proteins quite
AND
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well, as might be expected based on the discussion above, but the linearity in PEG concentration difference was not retained for higher molecular weight proteins. Extending [2] to second order in (wi - w!$ allowed the equation to fit data for a variety of proteins at pH 7.0, regardless of their isoelectric points (13). The temperature dependence expected from a literal interpretation of [l] (xij is proportional to l/T) was also found to hold. Hence, the polymer concentration dependence of the protein K seems well established. The above correlation is useful but the predictive powers of Eq. [2] are limited since the constants must be measured for every phase system, i.e., for each polymer type and molecular weight, buffer type and concentration, etc. While [l] provides expressions for the constants, there are fundamental objections to its literal application, as we have seen. The osmotic virial coefficient approach or its variants are designed to alleviate some of these difficulties by providing a framework with which to extend a limited number of measurements on two- or three-component solutions to the prediction of all the thermodynamic properties of the systems. This includes, of course, predictions of the KS of added proteins and, in principle, the effects of such additions on the properties of the phase systems. With the objective of predicting protein partition effects, three groups have utilized the virial expansion method, the two groups mentioned above and Hall’s group (14). There are some fundamental differences in their approaches. Cabezas et al. (7,8,21) and Forciniti and Hall (14) begin with a constant pressure theory initiated by Hill (27) while King et al. (9) and Haynes et al. (10) extend the constant volume equations of Edmond and Ogston (20). The two methods are shown to produce equivalent equations for noninteracting solvents (14) but there can be significant differences in the coefficients under some con,ditions (Cabezas, submitted for publication) and the constant pressure approach seems the more realistic. The basic equation, for isoelectric proteins in the absence of salts, can be expressed as In K = A,(m!jj
- m$ + A,,(rni
- m”,)
[31
where mi is the molality of component i in the indicated phase and A,, and A,, are the virial coefficients to be evaluated. Clearly, this is of the same form as [l] and [2] above. A more complicated form results when salts are included and a major problem is to treat their interactions with the other components correctly. The virial coefficients which describe the binary interactions between each pair of components can be calculated from first principles (14), measured (9,lO) or obtained by a combination of these approaches. For example, molecular weight effects can be predicted given a value for a monomer interaction coefficient (8). Forciniti and Hall (14) have calculated the A, and A,, values
RECENT
RESULTS
IN
AQUEOUS
for PEG, Dx, and protein assuming there are no net attractions between any of the species. Their interactions simply consist of the repulsion associated with the assumption that no two molecules can occupy the same location. The coefficients can then be calculated from the volume from which the centers of the two interacting molecules are excluded, which depends on the geometry assumed for the two species. This was the original approach taken by Ogston (20) and has been applied in studies of protein/polymer mixtures (28,29). Forciniti and Hall calculated excluded volumes assuming the protein was a rigid sphere and the polymers behaved as spheres, rods, or Gaussian coils. None of these assumptions produced satisfactory fits to their data set, however, and they concluded that attractive interactions among some of the components must be included to reproduce the experimental results. In order to test their theoretical predictions, Forciniti, Hall, and Kula [ (30) and D. Forciniti, personal communication] amassed an enormous data set based on four Dx fractions and four PEG fractions which were combined at four compositions each at three temperatures and four proteins partitioned in each system at their isoelectric point and three other pH values (the pH dependence was studied only at one temperature). Molecular weight distributions were measured for each polymer fraction both in pure solution and in the separated phases of a number of systems as well. Once this information is published it will provide a valuable resource for comparison with theoretical predictions. The Berkeley group has recently utilized a version of Eq. [3] which includes salt interaction terms and concentrations and measures the virial coefficients as mentioned above, with the ion-related constants provided by membrane osmometry and published vapor pressure data (10). When salts which partition unequally between the phases, such as SO;’ or H,PO;, are present an electrostatic potential difference should appear between the phases and its contribution to the distribution of charged proteins has to be included in the calculation. ‘To date, computation of the partition behavior of bovine serum albumin as a function of tie line length in Dx-PEG systems containing either NaCl (which does not partition strongly in these systems) or NaH,PO, provides predictions which show the measured trends but are significantly higher than the measured KS. The discrepancy is felt to be due to neglect of ion pairing, inappropriate treatment of protein electrostatics, and neglect of higher order terms in the virial expansion, necessary to describe the phases at high tie line length (10). There are clearly many challenges to be taken up by those following the above approach. Salt-single polymer phase systems have received relatively little attention, for instance, although some interesting correlations of K with hydrophobicity (31) and free volume (32,33)
PHASE
PARTITIONING
5
have been made. Prediction of phase diagrams and protein KS for such systems will test the ability of the theory to handle concentrated salt solutions. Proteinprotein interactions have not yet been addressed (except among molecules of the same substance where the effect is likely to be small since K is usually independent of protein concentration). It should be possible to treat the protein-protein-water two-phase systems which have been reported (34) or protein-polymer-water systems (20,35) to investigate high protein concentration behavior. It will also be of interest to see whether parameters can be identified which correlate with the hydrophobicity of distributed material. Zaslavsky and collaborators have made an extensive investigation of this issue (36,37). While there is much to be gained by using the virial expansion approach to predict overall system behavior, by measuring rather than calculating key coefficients the physical picture of what is occurring is deemphasized. A series of investigations which is richer in this regard has originated from MIT (11,12,38) in which the lattice model is modified for a spherical protein interacting with a polymer phase. The issue of whether attraction between the protein and polymer is necessary to describe observed results was addressed by adapting the computationally intensive polymer adsorption theory of Scheutjens and Fleer (39) and calculating the free energy of the particle in each phase, the difference being proportional to In K. The treatment provides as well the polymer segment distribution profile in the vicinity of the protein. The protein-polymer segment interaction is described by x8, equal to the difference in the protein-solvent and protein-polymer dimensionless interaction energies per lattice site. It was found that a small attraction between protein and either polymer was necessary to reproduce experimental values for K as a function of PEG molecular weight. The required attraction is small, however, and the polymer is at a lower concentration near the surface of the protein than in the bulk due to the unfavorable entropy loss suffered by the polymer chains when their configurations are constricted by the surface. The overall free energy of the protein is therefore increased. That is, the protein is incompatible with both polymers, the exclusion being least for the polymer which predominates in the phase to which the protein partitions. It was found necessary to make the Dx-protein x8 slightly more attractive than the PEGprotein parameter. A similar conclusion was reached by Forciniti and Hall (14) in attempting to rationalize their data on the temperature dependence of K. This theory was further tested on data generated in polyvinyl methyl ether-PEG systems for a number of proteins and, with appropriate choice of parameters, reasonable agreement was achieved (38). Hence, there is reason to accept the general picture provided by these
6
WALTER,
JOHANSSON,
calculations. The problem with applying it in a more general sense is the difficulty in estimating some of the important parameters, particularly xs, independently. The lattice model also suffers from most of the same limitations with respect to solvent interactions as were discussed above for the Flory-Huggins calculations. A further contribution from the MIT group (1540) discusses the physical picture of protein-PEG interactions in the context of the scaling theory of polymer solutions initiated by de Gennes (41). For low molecular weight fractions (
BIOCHEMISTRY
197,
l-18
(19%)
REVIEW Partitioning in Aqueous Two-Phase Systems: Recent Results Harry Walter,* G&e Johansson,? and Donald E. Brooks$ *Laboratory of Chemical Biology, Veterans Affairs Medical Center, Long Beach, California 90822; TDepartment Chemical Center, University of Lund, S-221 00 Lund, Sweden; and SDepartments of Pathology and Chemistry, University of British Columbia, Vancouver V6T 1 W5, British Columbia, Canada
From analytical to commercial scale, aqueous twophase systems have a niche in the purification, characterization, and study of biomaterials (1). Initially described by Beijerinck toward the end of last century, such phase systems were rediscovered and first employed in the 1950s by Albertsson for partitioning biomaterials (2,3). Aqueous two-phase systems are generally composed of a water solution of two structurally distinct hydrophilic polymers or of one polymer and certain salts (e.g., alkali phosphates). Above critical concentrations of these components, spontaneous phase separation takes place with each of the two resulting phases enriched with respect to one of the components. The replacement of organic by aqueous solutions in immiscible two-phase systems has permitted the application of the classical separatory method of resolving components of a mixture by partitioning not only to labile macromolecules but also to cells, membranes, and organelles. Two-polymer aqueous phase systems can be buffered and, if necessary, rendered isotonic. Appropriate selection of polymers [the most widely used being dextran (Dx)’ and polyethylene glycol (PEG)] results in
i Abbreviations used: Ab, antibody; CCD, countercurrent distribution; Cross-point (isoelectric point), obtained by cross-partitioning, i.e., the pH at which two plots of Kvs pH for a material intersect when the K’s are measured in phase systems containing different salts but the same polymer concentrations; DEAE-, diethylaminoethyl-; DMSO, dimethylsulfoxide; Dx, dextran; EHEC, ethylhydroxyethylcellulose; FACS, fluorescence-activated cell sorter; FA-PEG, polyethylene glycol fatty acid esters; Fi, Ficoll; G, apparent partition coefficient (or ratio), obtained from the location of the peak in a CCD curve = r-l(n - rma.), where r,, is the tube (or cavity) number of the peak of the distribution and n is the number of transfers; HPD, hydroxypropyldextran; HPS, hydroxypropylstarch; IDA, iminodiacetic acid; IO, inside out; K, partition coefficient (used with materials that partition between the two bulk phases) = concentration of material in top phase/concentration of material in bottom phase; P, partition ratio (used with materials, e.g., cells, which partition between a bulk phase 0003-2697191 $3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
of Biochemistry,
phase systems which are mild and nondeleterious to most biomaterials partitioned in them. These phases have interfacial tensions several orders of magnitude lower than those of aqueous-organic or organic-organic systems. The manipulation of polymer and ion composition and concentration determines the physical properties of the phases which, in turn, effect separations of biomaterials based on different physical parameters. For example, even though Dx and PEG are themselves nonionic, certain salts (e.g., phosphates, sulfates) have different affinities for the two phases, a phenomenon which gives rise to a Donnan potential between the phases and causes them to be charge-sensitive. Other salts have equal affinities for the two phases and give rise to non-charge-sensitive systems. The incorporation of a ligand bound to one of the phase-forming polymers (or one which partitions or is made to partition extremely into one of the phases) permits affinity partitioning to be carried out. Partitioning of biological particulates has proved to be an extremely sensitive method for their separation and fractionation. The partitioning of larger particulates depends predominantly on their surface properties. By carrying out multiple extractions [e.g., countercurrent distribution (CCD)] even subtle surface alterations of cells that accompany normal and abnormal in uiuo processes (e.g., differentiation, maturation, aging, metastatic potential) or in vitro treatments can be traced as these are often reflected in altered partition ratios, i.e., P values (l-3). Membranes and organelles can be fractionated, plasma membranes can be purified, and membrane domains charted. Right-side out and inside out vesicles can be separated. Proteins, including isoenzymes, have been efficiently segregated while afand the interface) = quantity of material in bulk phase as a percentage of total material added, PS, photosystem; PVA, polyvinyl alcohol; PVP, polyvinylpyrrolidone; RO, right-side out; TMA-, trimethylamino. 1
Inc. reserved.
2
WALTER,
JOHANSSON,
finity partitioning has permitted the specific extraction of a multitude of proteins including dehydrogenases and kinases and the fractionation of nucleic acids on the basis of base- and sequence-specificity (l-4). The aqueous phases obtained with one polymer, usually PEG, and certain salts, which are not isotonic, have found their main use in the rapidly expanding biotechnology of protein purification (4). Pa~icularly advantageous is the fact that extractions can easily be scaled up. Because the high salt concentration of such systems often precludes affinity partitioning and because of the prohibitively high cost of dextran for commercial applications, other polymers (i.e., Dx substitutes) or polymer combinations are being examined for use in two-polymer aqueous phases. Other biotechnological applications of aqueous two-phase systems include extractive bioconversion processes and the concentration of biomaterials such as viruses. Since the parameters that determine the partition coefficient, I( (or the partition ratio, P), of materials are exponentially related to the K (or P) value, partitioning often yields separations and information on physical properties of biomaterials not readily obtained by other means. Here we relate some of the rapid advances in and novel applications of aqueous phase pa~itioning during the past 5 years, i.e., since the previous review (l), as well as theoretical aspects of phase separation and partitioning. It is gratifying that some of the “prospects” we noted (1) have come to pass. These include: finding inexpensive polymers with favorable properties, development of biospecific extraction methods (also for cells), new apparatuses for more rapid processing of materials, use of highly efficient columns for protein (and nucleic acid) fractionation based on two-polymer aqueous phases, development of systems for use at high and low temperatures, addition of small quantities of water-soluble organic solvents to aqueous phase systems to permit partitioning of materials with low solubility in water, and use of systems composed of more than two phases. THEORY
OF PETITIONING
PHENOMENA
Considerable advances in the theory of two-phase systems and partitioning phenomena have been made since this subject was last treated (5). These advances derive largely from the impetus provided by the potential of partitioning in biotechnology, particularly as one or more steps in the medium to large scale isolations of genetically engineered proteins. The chemical engineering community has been dealing with liquid-liquid extraction problems for decades (6) and has found in aqueous phase partitioning a fertile area for investigation. Experience in the industrial application of extraction has shown clearly that deeper understanding leads
AND
BROOKS
to more efficient separations and more profitable processes. The goal of many of the groups involved is to secure a sufficient understanding of macromolecular partitioning to allow prediction of K for a defined material under arbitrary conditions and hence to allow optimization of separation. In some of the approaches currently being pursued achievement of this goal implies an ability to predict phase behavior of the systems, i.e., to predict phase diagrams, on the basis of a minimum number of experimental measurements of characteristic constants describing interactions between pairs of elements in the system (7-10). Other groups have assumed the presence of an invariant two-phase system and concentrated on modeling the interactions between phase components which result in the observed K (11-14). In all cases to be reviewed significant agreement with experiment exists under some sets of conditions. However, no single approach has proven so superior that all would accept its universality. Due to space li~tations we will provide only a broad overview of the theoretical developments since 1985. For a more detailed discussion the excellent recent review by Abbott et al. (15) is recommended. Prediction of Phase
Diagrams
A sufficiently detailed understanding of the interactions which cause phase separation would obviously contribute greatly to our capacity to predict the K of added material, particularly if the partitioned material was a low molecular weight species or another polymer. Two approaches to the prediction of phase diagrams have been taken, one based on polymer solution theory and the other adapted from the thermodynamic treatments of liquid phase equilibria. In neither case have predictions of phase behavior been made from first principles, i.e., simply from the chemical structures of the components; liquid state theories are not available to describe such complex systems. Rather, various theoretical constructs are applied to sets of experimental data to evaluate the constants necessary to allow prediction of phase diagrams of related systems. Generally, the goal is to maximize predictive power with the minimum number of measured constants. The best known polymer solution treatment is the lattice theory of Flory and Huggins (5) which has been quite successful in predicting general properties of polymer solutions in unstructured solvents. In such models the solution is represented by a three-~mensional lattice, each site of which is filled with a solvent molecule or a segment of one of the polymers present. The free energy of mixing of the solution is evaluated from the sum of the energies of interaction between the various segments and solvents and from the configurational entropy associated with the numbers of ways of arranging the components on the lattice. If in a two-polymer, one-
RECENT
RESULTS
IN
AQUEOUS
solvent mixture the interaction energy between unlike polymer segments is even modestly unfavorable, the total free energy can be lowered-at the cost of an entropy decrease-if the number of such contacts is minimized by the formation of two phases, each rich in one of the polymers (5). In this lattice model the parameters which describe the system are the molar volumes of the polymers relative to the solvent molar volume, proportional to polymer molecular weights, and the interaction constants, xi j, which are proportional to the energy change which occurs on forming from pure components contacts between solvent (component 1) and either type of polymer segment (xl2 or x13) or contacts between the two-polymer segment types (xZ3). The derivation assumes the average polymer segment density is constant throughout the solution. Given the simplicity of the approach it has been remarkably successful at describing the thermodynamic properties of amorphous polymer mixtures and solutions. The main problem with applying this theory to aqueous polymer solution phase separation is that no solvent structuring effects are taken into account. While some ordering of solvent in contact with polymer can be introduced by allowing the interaction energy to contain a local entropy component the result is not well suited to strongly hydrogen bonding solvents like water. In fact, one whole class of phase separation phenomena, known as LCST behavior, cannot be described by the Flory-Huggins theory (16). This occurs when solutions of certain polymers such as PEG [or detergents with PEG headgroups (17,18)] are heated, reducing the Hbonding with water which holds them in solution. Nonetheless, the lattice theory formalism has been applied to fill out Dx-PEG-water phase diagrams using empirical values for the interaction parameters determined from one set of phase compositions and reasonable results were obtained (19). The values of the interaction parameters obtained do not appear to be applicable to other types of thermodynamic predictions, however [compare xij parameters with (ll)], which indicates the FloryHuggins theory is not accurately describing the details of the interactions present. An alternate approach is to apply what is known as the osmotic virial expansion in which thermodynamic functions, in particular the osmotic pressure or solvent chemical potential, are described by a power series in the polymer concentrations with empirically determined coefficients. The constants in the osmotic pressure expression which multiply the second order concentration terms, aij, are known as second virial coefficients and are related to the energies of interaction between the pairs of species represented by i and j. For instance, to describe two polymers in water the virial coefficients az2, us3, and az3 would have to be evaluated if the power series is taken to second order. The degree of compatibility or incompatibility between the
PHASE
3
PARTITIONING
polymers therefore is described by the sign and magnitude of uZ3. The method was first applied to describe phase separation of polymer (and protein) solutions by Edmond and Ogston (20) who found it could satisfactorily treat these systems. Recently, more rigorous calculations along the same lines have been made by groups in Berkeley (9,lO) and Arizona (7,B) as part of longer term efforts to predict protein partition. Both groups have taken the vital step of specifically including the effects of salts in their calculations. The two approaches differ in detail, particularly in the treatment of salt effects and polymer molecular weight. The Berkeley computation does not address the molecular weight issue and measures individual virial coefficients for each polymer fraction used. Cabezas et al. (7,8,21), on the other hand, have applied Renormalization Group ideas to allow calculations for all fractions of a given polymer whose molecular weights are known. Both groups use thermodynamic measurements (low angle light scattering, osmotic pressure, isopiestically determined activities) on two- or three-component solutions to obtain all the necessary constants; no adjustable parameters are utilized. Both have had considerable success at predicting phase diagrams for Dx-PEG (9,21), Dx-methylcellulose (21), and DxPEG-salt (8,lO) mixtures. Their most recent published work also includes predictions for the partition of salts in Dx-PEG systems which reproduce experimental data slightly less well than the phase diagrams. Predictions of PEG-salt phase diagrams are just becoming available (22). One unexpected prediction which should be tested experimentally is the dependence of the Dx-PEG phase diagram on very low concentrations of Na,SO, (8). Cabezas et al. claim that even 0.001% Na,SO, can shift the binodal to much lower Dx concentrations, arguing that experimentally no phase system is salt-free to this level so that true zero salt measurements have not been carried out. The published Berkeley calculations (9,lO) do not address this issue. Another area which deserves more consideration is the theoretical effect of polydispersity of the polymer fractions used, particularly the changes in molecular weight distribution which occur in the two phases relative to the source material. Some relevant theoretical work has appeared but it is difficult to apply it in the above formalisms (23,24). Some of the reported fractionation effects are dramatic (24). Prediction of Protein Partition
Coefficients
The first attempt at writing a molecular-level theory for partitioning of macromolecules in a two-polymer aqueous system again utilized the Flory-Huggins formalism, the partitioned material being treated as a third polymer (5). The result, obtained by equating chemical
4
WALTER,
JOHANSSON,
potentials of the partitioned species in the two phases and retaining only first order terms in the polymer concentration differences, provided an expression for the partition coefficient, K, in terms of the polymer concentration differences between the phases, the molecular weights of the two polymers and the added macromolecule and xij parameters for the macromolecule interacting with the solvent and each polymer: In K = PJ(cbl - d$)O - xlp) + (4: - &)(1/p,
- x2J
+ (4: - 4:) x (l/P, - x3p)l,
111
where
Pi = (molecular
volume of component i)l (molecular volume of solvent) i= 1, 2, 3, or p refers to solvent, polymer 1, polymer 2 or protein, respectively fraction of solution volume occupied by component i; t or b refer to top or bottom parameters describing the Xij = interaction i-j interaction, described above.
phase
Although it is clear that K describes the distribution of a flexible polymer coil, the predictions of the treatment also described most of the qualitative features observed for protein partition in the absence of electrostatic effects (25). That is, partition becomes more one-sided with increasing protein molecular weight and with increasing difference in concentration between the phases of either polymer. Partition also shifts in favor of a phase in which the polymer molecular weight is reduced, the more so the higher the protein molecular weight. The agreement with trends observed when polymer or protein molecular weights are changed may be fortuitous since the protein segments are neither uniformly distributed nor free to occupy large numbers of configurations, as assumed in the derivation. In the limit of low molecular weight proteins, i.e., for oligopeptides, the theory is much more realistic. A simplified version of the Flory-Huggins expression has been derived (26), utilizing the observation that in most Dx-PEG systems [but not in all two polymer mixtures (2)] the difference in Dx concentration between the phases is proportional to the difference in PEG concentrations. This reflects the fact that the tie lines in these systems are parallel in their phase diagrams. The equation is In K = A*(wi
- WE),
PI
where A* is an empirical parameter which depends on the molecular weights of the polymers and protein and w2 is the weight fraction of PEG in the indicated phase. Equation [2] was found to describe the partition of dipeptides and some low molecular weight proteins quite
AND
BROOKS
well, as might be expected based on the discussion above, but the linearity in PEG concentration difference was not retained for higher molecular weight proteins. Extending [2] to second order in (wi - w!$ allowed the equation to fit data for a variety of proteins at pH 7.0, regardless of their isoelectric points (13). The temperature dependence expected from a literal interpretation of [l] (xij is proportional to l/T) was also found to hold. Hence, the polymer concentration dependence of the protein K seems well established. The above correlation is useful but the predictive powers of Eq. [2] are limited since the constants must be measured for every phase system, i.e., for each polymer type and molecular weight, buffer type and concentration, etc. While [l] provides expressions for the constants, there are fundamental objections to its literal application, as we have seen. The osmotic virial coefficient approach or its variants are designed to alleviate some of these difficulties by providing a framework with which to extend a limited number of measurements on two- or three-component solutions to the prediction of all the thermodynamic properties of the systems. This includes, of course, predictions of the KS of added proteins and, in principle, the effects of such additions on the properties of the phase systems. With the objective of predicting protein partition effects, three groups have utilized the virial expansion method, the two groups mentioned above and Hall’s group (14). There are some fundamental differences in their approaches. Cabezas et al. (7,8,21) and Forciniti and Hall (14) begin with a constant pressure theory initiated by Hill (27) while King et al. (9) and Haynes et al. (10) extend the constant volume equations of Edmond and Ogston (20). The two methods are shown to produce equivalent equations for noninteracting solvents (14) but there can be significant differences in the coefficients under some con,ditions (Cabezas, submitted for publication) and the constant pressure approach seems the more realistic. The basic equation, for isoelectric proteins in the absence of salts, can be expressed as In K = A,(m!jj
- m$ + A,,(rni
- m”,)
[31
where mi is the molality of component i in the indicated phase and A,, and A,, are the virial coefficients to be evaluated. Clearly, this is of the same form as [l] and [2] above. A more complicated form results when salts are included and a major problem is to treat their interactions with the other components correctly. The virial coefficients which describe the binary interactions between each pair of components can be calculated from first principles (14), measured (9,lO) or obtained by a combination of these approaches. For example, molecular weight effects can be predicted given a value for a monomer interaction coefficient (8). Forciniti and Hall (14) have calculated the A, and A,, values
RECENT
RESULTS
IN
AQUEOUS
for PEG, Dx, and protein assuming there are no net attractions between any of the species. Their interactions simply consist of the repulsion associated with the assumption that no two molecules can occupy the same location. The coefficients can then be calculated from the volume from which the centers of the two interacting molecules are excluded, which depends on the geometry assumed for the two species. This was the original approach taken by Ogston (20) and has been applied in studies of protein/polymer mixtures (28,29). Forciniti and Hall calculated excluded volumes assuming the protein was a rigid sphere and the polymers behaved as spheres, rods, or Gaussian coils. None of these assumptions produced satisfactory fits to their data set, however, and they concluded that attractive interactions among some of the components must be included to reproduce the experimental results. In order to test their theoretical predictions, Forciniti, Hall, and Kula [ (30) and D. Forciniti, personal communication] amassed an enormous data set based on four Dx fractions and four PEG fractions which were combined at four compositions each at three temperatures and four proteins partitioned in each system at their isoelectric point and three other pH values (the pH dependence was studied only at one temperature). Molecular weight distributions were measured for each polymer fraction both in pure solution and in the separated phases of a number of systems as well. Once this information is published it will provide a valuable resource for comparison with theoretical predictions. The Berkeley group has recently utilized a version of Eq. [3] which includes salt interaction terms and concentrations and measures the virial coefficients as mentioned above, with the ion-related constants provided by membrane osmometry and published vapor pressure data (10). When salts which partition unequally between the phases, such as SO;’ or H,PO;, are present an electrostatic potential difference should appear between the phases and its contribution to the distribution of charged proteins has to be included in the calculation. ‘To date, computation of the partition behavior of bovine serum albumin as a function of tie line length in Dx-PEG systems containing either NaCl (which does not partition strongly in these systems) or NaH,PO, provides predictions which show the measured trends but are significantly higher than the measured KS. The discrepancy is felt to be due to neglect of ion pairing, inappropriate treatment of protein electrostatics, and neglect of higher order terms in the virial expansion, necessary to describe the phases at high tie line length (10). There are clearly many challenges to be taken up by those following the above approach. Salt-single polymer phase systems have received relatively little attention, for instance, although some interesting correlations of K with hydrophobicity (31) and free volume (32,33)
PHASE
PARTITIONING
5
have been made. Prediction of phase diagrams and protein KS for such systems will test the ability of the theory to handle concentrated salt solutions. Proteinprotein interactions have not yet been addressed (except among molecules of the same substance where the effect is likely to be small since K is usually independent of protein concentration). It should be possible to treat the protein-protein-water two-phase systems which have been reported (34) or protein-polymer-water systems (20,35) to investigate high protein concentration behavior. It will also be of interest to see whether parameters can be identified which correlate with the hydrophobicity of distributed material. Zaslavsky and collaborators have made an extensive investigation of this issue (36,37). While there is much to be gained by using the virial expansion approach to predict overall system behavior, by measuring rather than calculating key coefficients the physical picture of what is occurring is deemphasized. A series of investigations which is richer in this regard has originated from MIT (11,12,38) in which the lattice model is modified for a spherical protein interacting with a polymer phase. The issue of whether attraction between the protein and polymer is necessary to describe observed results was addressed by adapting the computationally intensive polymer adsorption theory of Scheutjens and Fleer (39) and calculating the free energy of the particle in each phase, the difference being proportional to In K. The treatment provides as well the polymer segment distribution profile in the vicinity of the protein. The protein-polymer segment interaction is described by x8, equal to the difference in the protein-solvent and protein-polymer dimensionless interaction energies per lattice site. It was found that a small attraction between protein and either polymer was necessary to reproduce experimental values for K as a function of PEG molecular weight. The required attraction is small, however, and the polymer is at a lower concentration near the surface of the protein than in the bulk due to the unfavorable entropy loss suffered by the polymer chains when their configurations are constricted by the surface. The overall free energy of the protein is therefore increased. That is, the protein is incompatible with both polymers, the exclusion being least for the polymer which predominates in the phase to which the protein partitions. It was found necessary to make the Dx-protein x8 slightly more attractive than the PEGprotein parameter. A similar conclusion was reached by Forciniti and Hall (14) in attempting to rationalize their data on the temperature dependence of K. This theory was further tested on data generated in polyvinyl methyl ether-PEG systems for a number of proteins and, with appropriate choice of parameters, reasonable agreement was achieved (38). Hence, there is reason to accept the general picture provided by these
6
WALTER,
JOHANSSON,
calculations. The problem with applying it in a more general sense is the difficulty in estimating some of the important parameters, particularly xs, independently. The lattice model also suffers from most of the same limitations with respect to solvent interactions as were discussed above for the Flory-Huggins calculations. A further contribution from the MIT group (1540) discusses the physical picture of protein-PEG interactions in the context of the scaling theory of polymer solutions initiated by de Gennes (41). For low molecular weight fractions (
