381
The molecular basis of partitioning in aqueous
lwo-phase systems Jonathan Huddleston, Andres Veide, Kristina K6hler, Julia Flanagan, Sven-Olof Enfors and Andrew Lyddiatt Protein purification based on partition in aqueous two-phase systems has attracted interest for many years. This approach has been advocated as a primary-stage unit operation in downstream processing. In reality, application has been strictly limited through inadequate understanding of the complex molecular forces involved in partitioning processes. Separations processes in biotechnology are defined by the nature of the product and its application. High degrees of purity which approach or attain molecular homogeneity may be required for some products, whereas simply the absence of conflicting activity is tolerated for others. Obtaining intracellular compounds as pure products requires a process comprising harvesting, product release, clarification, e~richment and fractionation. The early stages of such processes are characterized by attempts to maximize the product yield at the expense of retaining major contaminants. Usually, only then are high-resolution steps applied. Interest in exploiting aqueous twophase systems (Fig. 1) stems from their ability to combine several features of the early processing steps in only one or two partitioning operations. Thus, the technique has been devdoped as a primary purification step in which overall recovery, together with the removal of insolubles and major classes of contaminant, have been paramount. Further fractionation has been confined to conventional high-resolution techniques t. The advantages of the technique 1,2 include: • substituting difficult solid-liquid sepa~tions; • linearity of scale-up from the laboratory bench over several orders of magnitude; • rapidity using continuous mixers and centrifugal separators. Economic benefits over centrifugation and cross-flow filtration have been demonstra~:ed to scales involving 1000 kg biomass3. However, process-scale applications have not been widely exploited because mechanisms of partition are poorly understood and method development is wholly empirical. This con-
trasts unfavourably with conventional techniques of centrifugation, microfiltration, precipitation and adsorption. In addition, technical commercialization (so successful in the field of chromatography) has been hindered because the technique, as currently practiced, relies heavily on expertise rather than marketable hardware.
j. Huddleston, J. Fianagan and A. Lyddiatt are at the Biochemical Recovery Group, School of Chemical Engineering, University of Birmingham, Birmingham BI5 2TT, UK. A. Veide, K. K~hler and S-O. Enfors are at the Department of Biochemistry and Biotechnology, The Royal Instiaae of Technology, Stockholm, Sweden.
Partitioning of cell debris In process development, the effect of debris arising from cell disruption on partitioning characteristics of a system must be considered. The position of the
© 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/91~2.00
Process considerations and requirements To date, published processes4 that involve aqueous two-phase systems use several discrete equilibrium stages. This requires a simple batch-phase contact and separation at each stage and is normally carried out in centrifugal separators. Such operations were developed for efficient debris removal in recovery of intracellular proteins. Recently, the technique has been applied to the recovery of extracellu!ar proteins resulting in improved product concentration compared with other primary separationss. Primary extraction is arranged such that cell material and nucleic acids are collected in the denser lower phase, while the target protein partitions to the upper phase. A second extraction step may be applied to transfer the target protein into a fresh lower phase. This twostage cross-current extraction procedure can be automated and continuous 6. High-molecular-weight proteins may be recovered directly from the polyethylene glycol (PEG)-rich phase by ultrafiltration 7. Direct application of either phase to hydrophobic interaction matrices is possibles. A process scheme for the recovery ofl3-galactosidase from E. coli involving aqueous two-phase partition is shown in Fig. 2 (A. Veide, PhD thesis, Royal Institute of Technology, Stockholm, Sweden, 1987).
TIBTECHNOVEMBER1991 (VOL 9)
382
reviews
phase / "L / (PEG rich) / £ " / / / t. , " / / / ///.
'////~,
'/
,/,//,/, --
--
"=--_----___-
. - _~-~. = =---~!
_
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phase "~"-~ (phosphate
rich)
_ __---~,,
,
., , , / Lower
=---'
I I
I
t
-
/
/
/
20
10
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I
tl~lI
10
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20
% w/w potassium phosphate
Figure 1 Aqueous two.phase systems may form on mixing solutions of polymer and salt, usually polyethylene glycol (PEG)and potassium phosphate. All points above the binodal curve ABC form biphasic systems, whereas all points below are homogenous. All systems on the line AC have identical chemical compositions of top (PEG-rich) and bottom (salt-rich) phases defined by the points A and C, respectively. B represents a critical point on the curve where the system changes from mono- to biphasic. Systems D~, D2 and D3 differ in the ratio of top and bottom phases (R = VJVb). This can be shown to be equivalent to R = the length ratio CD/AC. Distribution of macromolecules is characterized by the partition coefficient (K) or the ratio of concentrations in top and bottom phases where K -Ct/C b.
binodal in the phase diagram (Fig. 1) is generally displaced toward lower concentrations of phase-forming components with increasing quantities of cell debris in the phase system2. Thus, in Fig. 1, the curve delineating the boundary between mono- and biphasic systems is shifted towards the origin when they are loaded with cell debris. This behaviour may also influence the recovery of target proteins in the extraction step, as described for protein-A-13-galactosidase fusions'~. Furthermore the desired one-sided distribution of cell debris may be disturbed 2. This displacement of the binodal curve, for which cellular DNA seems primarily responsible, may be compenTIBTECHNOVEMBER1991(VOL9)
sated by reducing the amount of phase-forming polymers or salts used to form the system9. In partition of extracts of disrupted yeasd o, the overall recoveries of protein are reduced because the presence of cell debris in PEG-rich phases dictates use of only a discrete area of the phase diagram (e.g. within an area bounded by ABD 2 in Fig. i).
Partitioning of target protein Initial stages of downstream processing in product recovery must operate with high product yield. For a given partition coefficient K, desired yields can be achieved by adjusting the volume ratio of the two phases (top to bottom). However, since manipulating phase ratios in PECr-salt systems containing particulate material is confined to a discrete region of the phase diagram, the ability to manipulate yields is constrained by the requirement to partition cell debris to the lower phase 1°. Since the presence of biomass has a considerable effect on phase formation, a decrease in volume ratio is found with increasing biomass addition. To achieve 95% yields of particular products economically, a compromise between maximizing recovery and maximizing capacity may be necessary 1. Partitioning as a concentration step for extracellular proteins requires even higher values of K: e.g. >50 to achieve a threefold increase in concentration with similar 95% yields. It is therefore essential to be able to manipulate the partition coefficient in a controlled way, which presupposes the existence of a detailed understanding of the molecular mechanism of partition. Over the past 30 years, much empirical data has been assembled concerning the partition of macromolecules in PEG--dextran systems, and various thermodynamic descriptions of such systems are highly developed. However, fewer data are available regarding PEG--salt aqueous two-phase systems ! I, and little attention has been paid to determining the underlying physicochemical forces. This review therefore highlights limitations of our current understanding and outlines the need for a more rigorous investigation and efficient application of such systems. The basis of phase formation ~nd molecular separation Aqueous two-phase systems may form on mixing pairs or multiples of polymers in solution (polymerpolymer system) or between mixtures of polymers and salts (polymer--salt system). There are similarities and differences between these two principal forms of aqueous two-phase systems.
Phase separation ia polFmev-polFmersystems Many pairs of polymers, when mixed together in polar solvents above defined concentrations, separate into biphasic systems, with each phase preferentially enriched in one or other of the polymers. Few polymer mixtures have been rigorously investigated for the partition of biological macromolecules. Systems composed of PEG and dextran, or PEG and
383
reviews potassium phosphate, are the best known, though others (Table 1) have also been tried. Thermodynamically, phase separation can be described in terms of the requirement for positive free energy of mixing. This is due to the large enthalpy of mixing associated with the long polymer chains overcoming the loss of entropy inherent in segregation to different phases. Such a description has been the basis of several thermodynamic models of phase separation 1-', many of which can accurately reproduce phase diagrams and some aspects of macromolecular partition. However, no account is taken of the role of the solvent in the interactions involved. In aqueous systems, the polymer molecules are strongly hydrogen bonded to surrounding water molecules. At the low molecular masses common in bioseparations (
The molecular basis of partitioning in aqueous
lwo-phase systems Jonathan Huddleston, Andres Veide, Kristina K6hler, Julia Flanagan, Sven-Olof Enfors and Andrew Lyddiatt Protein purification based on partition in aqueous two-phase systems has attracted interest for many years. This approach has been advocated as a primary-stage unit operation in downstream processing. In reality, application has been strictly limited through inadequate understanding of the complex molecular forces involved in partitioning processes. Separations processes in biotechnology are defined by the nature of the product and its application. High degrees of purity which approach or attain molecular homogeneity may be required for some products, whereas simply the absence of conflicting activity is tolerated for others. Obtaining intracellular compounds as pure products requires a process comprising harvesting, product release, clarification, e~richment and fractionation. The early stages of such processes are characterized by attempts to maximize the product yield at the expense of retaining major contaminants. Usually, only then are high-resolution steps applied. Interest in exploiting aqueous twophase systems (Fig. 1) stems from their ability to combine several features of the early processing steps in only one or two partitioning operations. Thus, the technique has been devdoped as a primary purification step in which overall recovery, together with the removal of insolubles and major classes of contaminant, have been paramount. Further fractionation has been confined to conventional high-resolution techniques t. The advantages of the technique 1,2 include: • substituting difficult solid-liquid sepa~tions; • linearity of scale-up from the laboratory bench over several orders of magnitude; • rapidity using continuous mixers and centrifugal separators. Economic benefits over centrifugation and cross-flow filtration have been demonstra~:ed to scales involving 1000 kg biomass3. However, process-scale applications have not been widely exploited because mechanisms of partition are poorly understood and method development is wholly empirical. This con-
trasts unfavourably with conventional techniques of centrifugation, microfiltration, precipitation and adsorption. In addition, technical commercialization (so successful in the field of chromatography) has been hindered because the technique, as currently practiced, relies heavily on expertise rather than marketable hardware.
j. Huddleston, J. Fianagan and A. Lyddiatt are at the Biochemical Recovery Group, School of Chemical Engineering, University of Birmingham, Birmingham BI5 2TT, UK. A. Veide, K. K~hler and S-O. Enfors are at the Department of Biochemistry and Biotechnology, The Royal Instiaae of Technology, Stockholm, Sweden.
Partitioning of cell debris In process development, the effect of debris arising from cell disruption on partitioning characteristics of a system must be considered. The position of the
© 1991, Elsevier Science Publishers Ltd (UK) 0167 - 9430/91~2.00
Process considerations and requirements To date, published processes4 that involve aqueous two-phase systems use several discrete equilibrium stages. This requires a simple batch-phase contact and separation at each stage and is normally carried out in centrifugal separators. Such operations were developed for efficient debris removal in recovery of intracellular proteins. Recently, the technique has been applied to the recovery of extracellu!ar proteins resulting in improved product concentration compared with other primary separationss. Primary extraction is arranged such that cell material and nucleic acids are collected in the denser lower phase, while the target protein partitions to the upper phase. A second extraction step may be applied to transfer the target protein into a fresh lower phase. This twostage cross-current extraction procedure can be automated and continuous 6. High-molecular-weight proteins may be recovered directly from the polyethylene glycol (PEG)-rich phase by ultrafiltration 7. Direct application of either phase to hydrophobic interaction matrices is possibles. A process scheme for the recovery ofl3-galactosidase from E. coli involving aqueous two-phase partition is shown in Fig. 2 (A. Veide, PhD thesis, Royal Institute of Technology, Stockholm, Sweden, 1987).
TIBTECHNOVEMBER1991 (VOL 9)
382
reviews
phase / "L / (PEG rich) / £ " / / / t. , " / / / ///.
'////~,
'/
,/,//,/, --
--
"=--_----___-
. - _~-~. = =---~!
_
:___ -
_
---~'--~, ~__~___~_
--
phase "~"-~ (phosphate
rich)
_ __---~,,
,
., , , / Lower
=---'
I I
I
t
-
/
/
/
20
10
/
I
tl~lI
10
/
~
:
/
/
/
\',,
_--_..
=" - - ----------: .....
/
/
/
/
20
% w/w potassium phosphate
Figure 1 Aqueous two.phase systems may form on mixing solutions of polymer and salt, usually polyethylene glycol (PEG)and potassium phosphate. All points above the binodal curve ABC form biphasic systems, whereas all points below are homogenous. All systems on the line AC have identical chemical compositions of top (PEG-rich) and bottom (salt-rich) phases defined by the points A and C, respectively. B represents a critical point on the curve where the system changes from mono- to biphasic. Systems D~, D2 and D3 differ in the ratio of top and bottom phases (R = VJVb). This can be shown to be equivalent to R = the length ratio CD/AC. Distribution of macromolecules is characterized by the partition coefficient (K) or the ratio of concentrations in top and bottom phases where K -Ct/C b.
binodal in the phase diagram (Fig. 1) is generally displaced toward lower concentrations of phase-forming components with increasing quantities of cell debris in the phase system2. Thus, in Fig. 1, the curve delineating the boundary between mono- and biphasic systems is shifted towards the origin when they are loaded with cell debris. This behaviour may also influence the recovery of target proteins in the extraction step, as described for protein-A-13-galactosidase fusions'~. Furthermore the desired one-sided distribution of cell debris may be disturbed 2. This displacement of the binodal curve, for which cellular DNA seems primarily responsible, may be compenTIBTECHNOVEMBER1991(VOL9)
sated by reducing the amount of phase-forming polymers or salts used to form the system9. In partition of extracts of disrupted yeasd o, the overall recoveries of protein are reduced because the presence of cell debris in PEG-rich phases dictates use of only a discrete area of the phase diagram (e.g. within an area bounded by ABD 2 in Fig. i).
Partitioning of target protein Initial stages of downstream processing in product recovery must operate with high product yield. For a given partition coefficient K, desired yields can be achieved by adjusting the volume ratio of the two phases (top to bottom). However, since manipulating phase ratios in PECr-salt systems containing particulate material is confined to a discrete region of the phase diagram, the ability to manipulate yields is constrained by the requirement to partition cell debris to the lower phase 1°. Since the presence of biomass has a considerable effect on phase formation, a decrease in volume ratio is found with increasing biomass addition. To achieve 95% yields of particular products economically, a compromise between maximizing recovery and maximizing capacity may be necessary 1. Partitioning as a concentration step for extracellular proteins requires even higher values of K: e.g. >50 to achieve a threefold increase in concentration with similar 95% yields. It is therefore essential to be able to manipulate the partition coefficient in a controlled way, which presupposes the existence of a detailed understanding of the molecular mechanism of partition. Over the past 30 years, much empirical data has been assembled concerning the partition of macromolecules in PEG--dextran systems, and various thermodynamic descriptions of such systems are highly developed. However, fewer data are available regarding PEG--salt aqueous two-phase systems ! I, and little attention has been paid to determining the underlying physicochemical forces. This review therefore highlights limitations of our current understanding and outlines the need for a more rigorous investigation and efficient application of such systems. The basis of phase formation ~nd molecular separation Aqueous two-phase systems may form on mixing pairs or multiples of polymers in solution (polymerpolymer system) or between mixtures of polymers and salts (polymer--salt system). There are similarities and differences between these two principal forms of aqueous two-phase systems.
Phase separation ia polFmev-polFmersystems Many pairs of polymers, when mixed together in polar solvents above defined concentrations, separate into biphasic systems, with each phase preferentially enriched in one or other of the polymers. Few polymer mixtures have been rigorously investigated for the partition of biological macromolecules. Systems composed of PEG and dextran, or PEG and
383
reviews potassium phosphate, are the best known, though others (Table 1) have also been tried. Thermodynamically, phase separation can be described in terms of the requirement for positive free energy of mixing. This is due to the large enthalpy of mixing associated with the long polymer chains overcoming the loss of entropy inherent in segregation to different phases. Such a description has been the basis of several thermodynamic models of phase separation 1-', many of which can accurately reproduce phase diagrams and some aspects of macromolecular partition. However, no account is taken of the role of the solvent in the interactions involved. In aqueous systems, the polymer molecules are strongly hydrogen bonded to surrounding water molecules. At the low molecular masses common in bioseparations (
