Biomolecule Separation Using Temperature-Induced Phase

Above the copolymer's cloud point, hydrophobic peptides will partition to the .... Separation of amino acids and small peptides with these systems are...
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Biomolecule Separation Using Temperature-Induced Phase Separation with Recycling of Phase-Forming Polymers Josefine Persson, Hans-Olof Johansson, and Folke Tjerneld* Department of Biochemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden

We review our recent work on the thermoseparating polymers in aqueous two-phase extractions of biomolecules. Random copolymers of ethylene oxide and propylene oxide (EOPO copolymers) are thermoseparating in water solution, i.e., at temperatures above the cloud point a concentrated copolymer phase and a water phase almost free of copolymer are formed. This phase behavior makes it possible to recycle copolymers after biomolecule extraction. A system for protein purification has been developed based on an EOPO copolymer/hydroxypropyl starch aqueous two-phase system. The target protein is extracted into the EOPO phase. Nonionic surfactants have been added to improve the partitioning to the copolymer phase. The EOPO phase is removed after extraction, and the temperature is increased above the cloud point. The target protein is recovered in the water phase, and the copolymer and surfactant is obtained as a concentrated phase free from protein. Copolymer and surfactant can be recovered after thermoseparation to 85-90% and four recycle steps have been accomplished. Covalent binding of an affinity ligand to the EOPO copolymer can improve the partitioning of a target protein to the EOPO phase. The EOPO ligand can be recycled after thermal separation. A novel one-polymer aqueous twophase system containing only thermoseparating EOPO copolymer and water has been developed for smaller biomolecules, e.g., peptides. Above the copolymer’s cloud point, hydrophobic peptides will partition to the EOPO-enriched phase and hydrophilic peptides to the water phase. Proteins can be extracted in a one-polymer phase system containing a hydrophobically modified EOPO copolymer (HM-EOPO). The target protein is partitioned to the HM-EOPO phase. The copolymer can be recycled after back-extraction of the protein to a new water phase. Introduction Aqueous two-phase systems have gained a relatively widespread use for separation of biological materials [Albertsson, 1986; Walter et al., 1985; Walter and Johansson, 1994]. Recent applications have also been on extractions of metal ions [Rogers et al., 1996]. The most well-known aqueous phase systems are the two polymer systems, e.g. poly(ethylene glycol) (PEG)/dextran, and the system with PEG and salt, e.g. PEG/ phosphate. The two-phase systems obtained after phase separation have high water content in both phases, typically 80-90%, and this makes them especially suitable for separations of sensitive biological materials. The PEG/dextran system is used in lab-scale separations in biochemistry and cell biology, e.g., for separations of membrane fractions, cell organelles, and whole cells. The PEG/salt systems are based on inexpensive chemical compounds and have found industrial use for large-scale purification of proteins [Hustedt et al., 1985; Woker et al., 1994]. The PEG/salt systems have also been found to be suitable for metal extractions [Rogers et al., 1996]. In this paper we will review our recent work on the use of thermoseparating polymers in aqueous two-phase systems, which has been a new development in aqueous two-phase extractions. Two aqueous phases can be formed by heating an aqueous solution of a thermoseparating polymer above a critical temperature, the cloud point [Alred et al., 1993, 1994; Harris et al., 1991; Johansson et al., 1993, 1995, 1996, 1997a,b; Persson et * Corresponding author. Fax: +46 46 222 45 34. E-mail: [email protected].

al. 1998, 1999a,b]. One of the phases (often the bottom phase) is enriched with polymer, the other is depleted. The lowest cloud point of the system is the lower critical solution temperature, LCST. Thermoseparation is not a common phenomenon since most molecules have an increased solubility upon temperature increase. For small molecules it is very rare. A well-known exception is nicotine in water [Hudson, 1904; Shinoda, 1978]. Examples of polymers which display thermoseparation in water are random or block copolymers of ethylene oxide (EO) and propylene oxide (PO), poly(vinylcaprolactam), poly(N-isopropylacrylamide), and ethyl-hydroxyethylcellulose (EHEC). A review of polymers which are thermosensitive and with applications in biotechnology has been made by Galaev and Mattiasson [1993]. Some of these polymers (e.g., EHEC) form stiff gels upon temperature increase [Johansson et al., 1993, 1997a]. The use of such polymer systems for separation processes has been investigated little. The random copolymers of EO and PO segments (henceforth called EOPO copolymers) form a liquid polymer-rich phase in equilibrium with a water phase upon temperature increase. Most studies of biomolecule partitioning in thermoseparated systems has been performed in EOPO-copolymercontaining systems. In these systems the concentration of EOPO-copolymer in the bottom phase is usually 4060%, while the top phase contains almost 100% water. EOPO copolymers used have had compositions from 20% EO, 80% PO to 50% each of EO and PO (Ucon 50 HB 5100 (Ucon) and Breox 50A1000 (Breox)). By use of thermoseparating polymers, it has been possible to combine two-phase extractions with temperature-induced phase separations. The systems used

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have been EOPO/dextran and EOPO/hydroxypropyl starch, where in each case the top phase polymer PEG was replaced with an EOPO copolymer. The hydroxypropyl starch is more cost efficient than dextran in large-scale extractions [Tjerneld et al., 1986]. A twostage separation process has been developed by this approach [Harris et al., 1991; Persson et al., 1999a]. The crude cell extract is introduced into the primary EOPO/ polysaccharide system. The phase composition is adjusted so that the target protein is extracted to the top EOPO phase. After separation of the phases and isolation of the EOPO phase in a separate vessel the temperature is increased over the EOPO copolymer cloud point. A new two-phase system is obtained where the target protein is recovered in the water phase. The EOPO copolymer phase is free from protein and can be recycled to a new aqueous two-phase system. Thus, several parts of this process are environmentally friendly. The target protein is separated from the polymer in the thermoseparation step, the top phase forming polymer can be recycled and the bottom phase forming polymer is a biodegradable starch polymer. Recently, an aqueous two-phase system composed of two thermoseparating polymers has been introduced [Persson et al., 1999b]. It is a system containing EOPO copolymer and a hydrophobically modified EOPO polymer (HM-EOPO). In this system either phase can be used for extraction of the target protein since both phases can be thermoseparated. After thermoseparation the protein can be recovered in a water phase and both of the polymers can be recycled. A water/EOPO two-phase system is formed above the cloud point in a water solution of an EOPO random copolymer. Proteins have in these systems been found to be excluded from the polymer-rich phase and partitioned to 100% to the water phase. The reason for this is that usually the surface of native proteins is too hydrophilic to be solubilized in the hydrophobic polymer rich bottom phase. There is also an entropic effect which arises from the large difference between the water phase and the polymer-rich phase in the number of molecules per volume (i.e., number density). This effect favors partitioning to the phase with highest number density (i.e., water phase) and is sometimes called the excluded volume effect [Johansson et al., 1998]. The protein partition to the water is desirable and forms the basis for the two-stage process described above. However, the water/EOPO two-phase system is interesting for separation of low molecular weight compounds where the excluded volume effects have much less importance. Separation of amino acids and small peptides with these systems are of special interest [Johansson et al., 1995; Johansson et al., 1997b]. Protein separation is possible in the recently introduced one-polymer two-phase system with HM-EOPO [Johansson et al., 1999]. Both phases in the water/HM-EOPO system contain low concentrations of polymer, e.g., 1% polymer in the water phase and 7% in the copolymer-enriched phase. Thus, the entropic effect is low in this system and proteins can be partitioned between both of the phases [Johansson et al., 1999]. For separating amphiphilic molecules such as amino acids and oligopeptides by a two-phase system, it is important to use a system with a suitable difference in hydrophobicity between the phases. If this difference is very large as in the octanol/water system, then all the components will be excluded from the hydrophobic

phase due to the low solubility of amino acids in octanol. If it is very small as in the PEG/dextran system then the low molecular weight components will partition almost evenly between the phases. For these types of components the water/EOPO system is more useful since the difference in hydrophobicity between the water phase and the polymer-rich phase is of intermediate character. The partitioning of charged molecules is strongly affected by the pH of the system and the addition of small amounts of salts. In the PEG/ phosphate system the partitioning of charged molecules is strongly affected by the phase-forming phosphate salt. Minor additions of other salts will not affect the partitioning in this system. In contrast to this, minor additions of salts or acids/bases in a water/EOPO is a useful way to control the partitioning of charged molecules. Also, the water/EOPO has the environmental advantage of not having phosphate and other salts as phase-forming agents. In this article we review the above-mentioned recent experimental work. For more complete details the reader is referred to Alred et al. [1994], Johansson et al. [1997b], and Persson et al. [1998, 1999a,b], and references therein. Materials and Methods Thermoseparating Polymers. EOPO random copolymers with different EO and PO compositions and molecular weights, e.g. EO30PO70, Mr 5000, were obtained from Shearwater Polymers (Huntsville, AL). The numbers after EO and PO indicate the weight percent of ethylene oxide and propylene oxide, respectively. The EOPO random copolymer Ucon 50-HB-5100 (Mr 4000) is produced by Union Carbide (New York, NY). It contains 50% (w/w) each of EO and PO (EO50PO50). Another nearly identical bulk polymer, Breox PAG 50A 1000 (also EO50PO50) (Mr 3900) was obtained from International Speciality Chemicals Ltd. (Southampton, England). The hydrophobically modified EOPO copolymer (HM-EOPO), Mr 8000, is produced by Akzo Nobel (Stenungsund, Sweden). This copolymer has an isophoronediisocyanate group in the center and on both sides of this group random copolymer chains of ethylene oxide and propylene oxide are attached. Each EOPO chain is composed of 66 EO and 14 PO groups and on the ends of the polymer aliphatic C14H29 groups are attached. Starch Derivatives. Starch derivatives that can be used as substitute for dextran in aqueous two-phase systems are the hydroxypropyl starches Reppal PES100 (Mr 100 000) and Reppal PES 200 (Mr 200 000) from Carbamyl AB (Kristianstad, Sweden) [Persson et al., 1998]. Two-Phase Systems. All polymer concentrations were calculated as % (w/w). The two-phase systems were prepared by dissolving the pure polymers in the protein solution obtained after cell removal and ultrafiltration. All experiments were performed in duplicate, and data are the average values. The 5 g systems were mixed with the help of a magnetic stirrer and the 5 kg system was mixed with a motor driven propeller. The separation into two phases was assisted by centrifugation at 1360g, 10 min. The partitioning of molecules in twophase systems is described by the partition coefficient K. It is defined as the concentration in the top phase, CT, of the molecule of interest divided by the concentration in the bottom phase, CB: K ) CT/CB.

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Temperature-Induced Phase Separation. The top phase of the primary phase system, containing EOPO copolymer, was removed and isolated in a separate vessel. The temperature of this phase was increased above the copolymer’s cloud point. For the EO50PO50 top phases a temperature of 60 °C was used, and for EO30PO70, 50 °C was used to achieve separation [Persson et al., 1998]. Apolipoprotein A-1M. Recombinant apolipoprotein A-1M was expressed in E. coli fermentations. Apo A-1M is exported to the periplasmic space and is subsequently obtained in the fermentation broth. The cells were removed by flocculation followed by centrifugation. The supernatant containing Apo A-1M was concentrated 10 times by ultrafiltration using a membrane with a 10 000 Mr cutoff. The concentration of Apo A-1M after ultrafiltration was 2.8 mg/mL and the total protein concentration was 30 mg/mL. The buffer in this starting material was 20 mM Tris-HCl pH 8, 150 mM NaCl, 10 mM EDTA, and 0.1% Tween 80. Determination of Protein and Apo A-1M. The total protein content was determined according to Bradford [Bradford, 1976], using Coomassie Brilliant Blue G. The absorption was measured at 595 and 465 nm, and the absorption at 465 nm was then subtracted from the 595 nm absorption. Bovine serum albumin was used for standard. The spectrophotometer used was UV-2101 PC from Shimadzu (Kyoto, Japan). The concentration of Apo A-1M was measured by an ELISA assay. An antibody, mouse IgG, recognizing Apo A-1M was coated on microtiter plates. Apo A-1M was bound to the primary antibody and a second antibody conjugate, biotin rabbit anti-mouse IgG, was bound to the complex. After washing, the complex was detected by adding ExtraAvidin with alkaline phosphatase conjugate. The alkaline phosphatase was used as marker and the absorption was measured at 405 nm. The yield was calculated as

yield ) (CtApoVt)/(CApoV) where CtApo is the concentration of Apo A-1M in the top phase and Vt is the volume of the top phase. CApo is the concentration of Apo A-1M in the starting material and V is the volume of material added to the system. The purification factor of Apo A-1M in the systems was calculated as t

t

purification factor ) (C Apo/C )/(CApo/C) where Ct is the protein concentration in the top phase and C is the protein concentration in the starting material. Samples from top phases were also analyzed with SDS-PAGE, 20% Phast gels (Amersham Pharmacia Biotech, Sweden) and stained with Coomassie R 350 (Phast Gel Blue R). After destaining, the gels were scanned with a densitometer (Personal Densitometer SI, Molecular Dynamics, Sunnyvale, CA) [Persson et al., 1998]. Phase Diagrams. The borderline between one- and two-phase regions is called the binodial curve. The polymer concentrations of the two phases in equilibrium are described by tie lines [Walter and Johansson, 1994]. The concentration of Reppal PES 100 was determined with polarimetry using a digital polarimeter (model AA10) from Optical Activity (London, U.K.). By making a

Figure 1. Experimentally determined phase diagram for the Ucon 50-HB5100/Reppal PES 100/water system. The phase diagram was determined at 20 °C. The region within the binodal curve is the two-phase region, representing conditions at which the polymer solution separates into two macroscopic phases. The region outside the binodal curve is the one-phase region, representing conditions at which the polymer solution exists as a single homogeneous phase (data from Persson et al., 1998).

polarimetric standard curve for Reppal PES 100 the specific rotation could be determined, 194.6 deg mL g-1 dm-1. The refractive index of Reppal PES 100 and Ucon was determined at 20 °C with a refractometer from Carl Zeiss (Oberkochen/Wu¨rtt., Germany). Different concentrations of Reppal PES 100 and Ucon were mixed and the resultant systems were separated into two phases by centrifugation for 10 min at 1360g. Samples from the top and bottom phases were diluted and measured with polarimetry and refractive index. The Reppal PES 100 concentration in both phases was determined by polarimetry. Ucon concentrations were determined by refractometry by subtracting the refractive index contribution of Reppal PES 100. Some points, near the critical point, were determined by titrating the two-phase system with water until a one-phase system was reached. For complete details of experimental procedures with affinity ligands in thermoseparating systems, see Alred et al. [1992], and for partitioning of amino acids and peptides in EOPO-water systems, see Johansson et al. [1997b]. Results and Discussion Phase Diagram for Polymer-Polymer Systems. In Figure 1 the phase diagram of Ucon 50-HB-5100/ Reppal PES 100 is shown. It is qualitatively similar to that of the PEG/dextran system. Because of the relatively low molecular weight of the starch polymer (100 000), when compared to dextran (500 000), higher concentrations are needed. The thermoseparating polymer Ucon 50-HB-5100 is enriched in the top phase which facilitates the removal of the Ucon phase after separation. Temperature-Induced Phase Separation. Thermoseparating polymers separate from water solutions above a certain temperature, the cloud point temperature or LCST [Saeki et al., 1976]. Depending on the type, the thermoseparated polymer may separate from the solution as a solid precipitate, a gel or a liquid phase [Galaev and Mattiasson, 1993]. The polymers which separate as a liquid phase are used preferentially, since in these cases the phases are better defined and more

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Figure 2. A temperature-concentration phase diagram for Ucon 50 HB-5100 (EO50PO50) (b) and HM-EOPO (9) in water. Twophase systems are formed above the binodal curve. Below this curve the solutions are a one-phase system (data from Persson et al., 1999b).

easily handled. Examples of polymers with this property are ethylene oxide-propylene oxide random copolymers. PEG which contains 100% ethylene oxide (EO) has a cloud point above 100 °C which is a too high temperature for many biomolecules. The higher the propylene oxide (PO) content the lower is the cloud point [Bailey and Callard, 1959; Louai et al., 1991a]. Solutions of 10% EO50PO50, EO30PO70 and EO20PO80 have cloud points of 50, 40,and 30 °C, respectively. A temperature concentration phase diagram for Ucon 50-HB-5100 (EO50PO50) in water and HM-EOPO in water is shown in Figure 2 [Johansson et al., 1993, 1999]. The cloud point is also affected by the molecular weight, for a larger polymer the cloud point is lower [Saeki et al., 1976]. The two-phase formation starts at the cloud point. However, two macroscopic phases are formed quickly (within less than 15 min) only if the temperature of the system is at least a few degrees above the cloud point. For a two-component polymer-water system the bottom phase is usually polymer-enriched, where the

EOPO copolymer concentration is about 40-60%. The top phase contains almost 100% water (see the phase diagram in Figure 2) [Johansson et al., 1993]. As shown in Figure 2 the HM-EOPO concentration in both phases after thermoseparation is relatively low, e.g., 1% in the water phase and 7-9% in the polymer-enriched phase. Temperature-Induced Separations of Proteins. The random EOPO copolymers have been used in aqueous two-phase systems for bioseparation. The systems studied have been EOPO/dextran and EOPO/ hydroxypropyl starch systems [Alred et al., 1993, 1994; Harris et al., 1991; Berggren et al., 1995; Johansson et al., 1996]. In these types of systems the EOPO copolymer is enriched in the top phase and the polysaccharide is enriched in the bottom phase. Target proteins can be partitioned to the top phase by utilizing salt effects or hydrophobic or affinity interactions [Alred et al., 1992; Berggren et al., 1995; Johansson et al., 1996]. The separation of the target protein from the EOPOcopolymer is accomplished by heating the top phase above the cloud point. This will induce a new two-phase system where the new top phase consists mainly of water and almost no polymer, and the new bottom phase is strongly enriched with polymer, see Figure 3. The target protein is completely excluded from the polymerrich phase. Thus, the thermoseparation property in this system is used to separate polymer from protein, and also to recover the polymer and reuse it in a new aqueous two-phase system. An EOPO/hydroxypropyl starch system was used to purify apolipoprotein A-1, an amphiphilic protein, from an E. coli fermentation solutions. The system was optimized for high recovery of apolipoprotein A-1 by addition of urea [Persson et al., 1998] and nonionic surfactants [Persson et al., 1999a]. Urea in high concentrations is often used to denature proteins [Creighton, 1993]. Addition of urea leads to breaking of hydrogen-bonding interactions. The effect of urea on Apo A-1M purification was studied by varying the urea concentration from 1.0 to 4.2 M in phase systems with high polymer concentration, 21% Reppal PES 100-10% EO30PO70. When urea was added to the two-phase

Figure 3. Scheme of the purification of a target protein, black dots, in a EOPO/hydroxypropyl starch system. The phase composition is adjusted so that the target protein is extracted to the top EOPO phase. After separation of the phases and isolation of the EOPO phase in a separate vessel, the temperature is increased over the EOPO copolymer cloud point. A new two-phase system is obtained where the target protein is recovered in the water phase. The EOPO copolymer phase is free from protein and can be recycled to a new aqueous two-phase system.

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Ind. Eng. Chem. Res., Vol. 39, No. 8, 2000 Table 1. Effects on the Purification and Yield of Apolipoprotein A-1M When Scaling up the Two-Phase System and the Temperature Induced Separationa purification factor

Figure 4. Effect on the degree of purification (9) and yield (%, b) of apolipoprotein A-1M in the primary top phase when urea was added to the two-phase system. Cell-free E. coli fermentation solution was added to the system to give a final protein concentration of 21 mg/mL. The phase systems had the polymer concentrations 21% Reppal PES 200-10% EO30PO70 (w/w). The data were calculated from ELISA results (data from Persson et al., 1998).

systems a better purification and yield was obtained (Figure 4) [Persson et al., 1998]. One explanation can be increased solubility of Apo A-1M in the EO30PO70 phase at higher urea concentration. The exposure of hydrophobic surfaces on Apo A-1M by partial unfolding in urea solution will lead to stronger interaction with the relatively hydrophobic EOPO copolymer, thus leading to reduced aggregation of Apo A-1M molecules and increased partitioning to the EOPO phase. An additional effect of urea will be the breaking of aggregates between Apo A-1M and other proteins in the solution. The results indicated that a concentration of 2.5 M urea was optimal (Figure 4). Urea partitioned nearly evenly between the two polymer phases of the primary EOPO/ starch system, K ) 0.9. The partitioning was the same, independent of the urea concentration in the system, i.e., 1-4 M. Aqueous two-phase systems can readily be scaled up [Hustedt et al., 1985]. The partitioning of protein is independent of the size of a system. As long as the phase concentrations in the systems are the same, the partitioning will also be the same. However, phase separation times will increase and may also show a dependence on the geometry of the separation vessel. The time of phase separation can be reduced by use of centrifugal separation [Hustedt at al., 1985]. The scale-up of Apo A-1M purification was studied. The phase system that was found optimal in the small scale experiments was scaled up 1000 times (Table 1). The large scale experiments were carried out directly after termination of an E. coli fermentation. After fermentation the cells were removed by flocculation and centrifugation. The phase components were added directly to the cell-free E. coli protein solution. The final concentrations were 17% Reppal PES 200, 12% EO30PO70, 2.5 M urea, 3.8 mg/mL total protein, and 0.4 mg/mL Apo A-1M. The polymers were dissolved by stirring, and the phases were separated by centrifugation. The top EO30PO70 phase was pumped to a separate vessel and the temperature increased to 50 °C. After 30 min, the water phase containing Apo A-1M could be removed. Data on purification and yield from the primary separation and the thermoseparation are shown in Table 1 and compared with data from a 5 g

starting material E. coli protein solution 5 g system top phase from primary system water phase after temperature-induced separation starting material E. coli protein solution 5000 g system top phase from primary system water phase after temperature-induced separation

1

yield (%)

V (L)

100

0.0029

2.5

79

2.5

77

1

100

2.7

82

2.7

81

0.0020

volume reduction

0.69

2.5

1.80

0.72

a Apo A-1 M was purified from a cell-free E. coli fermentation solution with total protein concentration of 3.8 mg/mL and Apo A-1M concentration of 0.4 mg/mL. The phase system composition was 17% Reppal PES 200, 12% EO30PO70 and 2.5 M urea. The data were calculated from ELISA determinations (data from Persson et al., 1998).

system. As is clear from this table the scale-up, by a factor of 1000, can be performed with no loss of purification and yield. Another advantage with the two-phase extraction is the possibility to isolate the target protein in a smaller phase and thus obtain a volume reduction. This is shown (Table 1) in the volume reduction factor which compares the volume of starting material and water phase after thermal separation. A volume reduction of 30% was obtained both in small and large scale. DNA Removal. The removal of DNA is of importance in processes which involve recombinant organisms. Therefore, DNA partitioning was investigated. E. coli DNA was partitioned strongly to the bottom phase in the primary aqueous two-phase system (10% EO30PO70-21% Reppal PES 200) with Tris buffer, pH 8.0, and 3.0 M urea. The partition coefficient in the system was 0.01. Thus, DNA was effectively separated from target protein in this aqueous two-phase extraction. The extreme partitioning of DNA agrees with results previously reported by Albertsson [1986]. Recycling of Phase Components. Nonionic surfactants with ethylene oxide groups (Triton X-100 or Tween 80) were added to the phase system to improve the partitioning of Apo A-1M [Persson et al., 1999a]. The surfactants were strongly partitioned to the top, EOPO, phase in an EOPO/hydroxypropyl starch system due to the ethylene oxide groups on the surfactant. The top phase in this case contained micelles of nonionic surfactants. Apolipoprotein A-1 has affinity to the surfactant micelles and was thus partitioned strongly to the top phase. The more hydrophilic bulk proteins could be partitioned to the bottom starch phase. When performing the thermoseparation both EOPO copolymer and the nonionic surfactant were partitioned to the copolymer phase, and the proteins were recovered in the water phase. In this way both copolymer and surfactant could be recovered and used in a new aqueous two-phase system. Polymer recycling was performed during repeated extractions of apolipoprotein A-1 from a cell-free E. coli fermentation solution [Persson et al., 1999a]. The recovered copolymer and surfactant were reused in three recyclings without loss in purification and yield of apolipoprotein A-1, see Figure 5. The purification factor was around 4.2-4.6 times, and

Ind. Eng. Chem. Res., Vol. 39, No. 8, 2000 2793 Table 2. Polymer Recovery by Thermoseparation. Recovery and Concentration of Polymers Were Determined in a Primary Protein-Free EO50PO50/ HM-EOPO System and in the Phases after Thermoseparationa recovery

concentration

EO50PO50 HM-EOPO EO50PO50 HM-EOPO (%) (%) (% w/w) (% w/w) EO50PO50/HM-EOPO EO50PO50 phase 98 HM-EOPO phase 2

5 95

13.9 0.2

0.3 11.8

2.3 52

0.1 2.1

Thermoseparation

Figure 5. Polymer recycling by thermoseparation. The purification factor and yield of apolipoprotein A-1Milano are given during extraction from an E. coli fermentation solution in the Breox/ hydroxypropyl starch system. The values are given for a system, 0, where new copolymer and surfactant was used and for systems where the copolymer and surfactant had been recycled one, two, and three times. Cell-free E. coli fermentation solution was added to the system to give a final protein concentration of 7 mg/mL. The composition of the primary system was 17% Breox 50A100012% Reppal PES 200 plus 1% Triton X-100. The buffer composition in the system was 14 mM Tris-HCl pH 8, 105 mM NaCl, 7 mM EDTA, and 0.07% Tween 80. The primary system was separated at 21 °C, and the thermoseparation was performed at 61 °C (data from Persson et al., 1999).

the yield was just under 80% in each recycling. These results were satisfactory because the apolipoprotein A-1 concentration in the original E. coli solution was about 10% of the total protein concentration. After the thermoseparation step 85-90% of the original copolymer and surfactant was recovered. The use of inexpensive starch polymers and recyclable EOPO copolymers makes this type of aqueous two-phase system a cost-efficient and environmentally favorable purification system. When using an aqueous two-phase system based on EOPO/HM-EOPO both of the polymers can be thermoseparated and recycled. The HM-EOPO polymer which has a lower cloud point and a higher molecular mass can be recovered at higher extent than the EOPO polymer (see Table 2), e.g., 97.5 and 73% respectively. Also in this system composed of two thermoseparating polymers the partition coefficient for protein and purification of apolipoprotein A-1 could be kept constant when recycling polymer [Persson et al., 1999b]. Recycling of Affinity Ligand by Thermoseparation. To improve the partitioning of a target protein to one of the phases in an aqueous two-phase system, affinity ligands to the target protein have been covalently attached to one of the polymer. Affinity partitioning in aqueous two-phase systems is extensively discussed by Walter and Johansson [1994]. A new way to recover the ligand-polymer has been studied by coupling the ligand to a thermoseparating EOPO random copolymer [Alred et al., 1992]. The ligand was a triazine dye, Procion Yellow HE-3G. The ligand has two reactive chlorine atoms where the end group of the random copolymer Ucon was added. Before the reaction the hydroxyl end group of Ucon was modified to an amino group to improve the reaction. For complete details of the Ucon modification see Alred et al. [1992]. The partition coefficient of glucose-6-phosphate dehydrogenase in a Ucon/dextran system, increased from 0.065 to 12 when ligand polymer was added (0.4% of total system). This system was exploited for purification of the enzyme from yeast homogenate and with a

EO50PO50 phase water phase polymer phase (EO50PO50) HM-EOPO phase water phase polymer phase (HM-EOPO) total polymer recovered

28 72 1 1 73

2 2.5 0 95

0.08 0.2

0.07 23.5

97.5

a The primary two-phase system contained 5% EO50PO50, 5% HM-EOPO and 10 mM sodium phosphate buffer pH 7.0 and was separated at 21 °C. The phases from the primary two-phase system were isolated in separate vessels and transferred to a water bath where the thermoseparation was performed at 55 °C for 30 min (data fron Persson et al., 1999b).

Figure 6. Enzyme purification scheme using affinity extraction of target enzyme with affinity ligand bound to Ucon. Temperatureinduced phase partitioning is used for enzyme recovery and recycling of Ucon 50-HB-5100 and Ucon-ligand. Table 3. Purification of Glucose-6-phosphate Dehydrogenase from Yeast Extracta sample

K (22 °C)

K (40 °C)

G6PDH Protein Ucon-PrY

12.4 0.32 24.6

>100 >100 0.32

purification factor (40 °C)

% recovered at 40 °C

4.2

78.8b 84.6c

a System is 6.3% Ucon 50-HB-5100, 9% dextran T40, 0.2% UconProcion yellow HE-3G, 0.02 M sodium phosphate buffer, pH 7.0 and 5.7% yeast extract. K value at 40 °C are for the partitioning between the water and Ucon phases formed by increase in temperature (data from Alred et al., 1992). b Recovered in water phase at 40 °C. c Recovered in Ucon phase at 40 °C.

subsequent separation of enzyme from ligand-polymer and recovery of the ligand polymer (see Figure 6). Glucose-6-phosphate dehydrogenase was purified from yeast extract in a Ucon/dextran aqueous two-phase system using 0.2% Ucon-Procion Yellow HE-3G (Table 3). In the first step the enzyme was separated from the bulk proteins. The enzyme was extracted by the Ucon ligand to the top phase (K ) 12) in the primary system. The bulk proteins were partitioned to the bottom phase (K ) 0.32). After phase separation the upper phase was withdrawn and isolated in a separate container. In the second step 0.2 M of Na2SO4 and 0.2 M NaCl were added

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to the withdrawn top phase to decrease the cloud point of the copolymer and to dissociate the enzyme from the Ucon ligand. The temperature was raised to 40 °C. In this step a new two-phase system which contained a Ucon-rich bottom phase and a water-salt-enriched top phase was found. In the new two-phase system the enzyme was recovered in the water phase with a yield of 79% and a purification factor of 4.2. The partition coefficient for the enzyme in the water/Ucon-phase system was >100. Ucon-Procion Yellow was recovered in the lower Ucon phase with a yield of 85%. No protein could be detected in this Ucon phase [Alred et al., 1992]. By using the thermoseparating property of the copolymer the target protein can be recovered in a water phase depleted from copolymer and the copolymer-ligand can be recovered and reused in a new aqueous two-phase system. Especially in the case of using expensive affinity ligands coupled to the copolymer it is of great interest to be able to recover the copolymer and ligand for further use. Thermal Separation in a One-Polymer System. The second type of system studied is a one-polymer aqueous two-phase system. The system is composed of a random EOPO copolymer with a concentration of 1020% (w/w) in water. By heating the system above the cloud point a two-phase system is formed, where the bottom phase is polymer enriched and the top phase is polymer depleted (see Figure 2). The addition of a third component to an EOPO copolymer-water system usually results in one of the following phenomena: (1) The cloud point is decreased, and the additive is strongly partitioned to the water phase. These effects are observed if the additive is very hydrophilic, e.g., salts [Ananthapadmanabhan and Goddard, 1987], glycine [Johansson et al., 1997a], and sugars [Sjo¨berg et al., 1989]. (2) The cloud point is decreased, and the additive is strongly partitioned to the polymer-rich phase. These effects are observed if the additive is hydrophobic, e.g., phenol, butyric acid [Johansson et al., 1993, 1997a; Louai et al., 1991b]. (3) The cloud point is almost unchanged. The additive has an almost even partitioning between the phases. In this case the additive has an intermediate chemical character in a hydrophilic-hydrophobic scale, e.g., acetic acid [Johansson et al., 1993] and ethanol [Louai et al., 1991b]. (4) The cloud point is increased or the system may lose the thermoseparating property. This effect can be obtained if the additive is a good solvent for the polymer and is highly concentrated in the system (e.g., acetic acid and butyric acid) [Johansson et al., 1993; Louai et al., 1991b]. It can also be obtained if the additive is strongly amphiphilic, e.g., sodium valerate [Johansson et al., 1997a] and SDS [Carlsson et al., 1988]. Addition of higher concentrations of urea and guanidinium hydrochloride also makes the polymer lose the thermoseparating property. Thus, by adding different cosolutes it is possible to manipulate several important properties of the aqueous EOPO copolymer system: cloud point, concentration of polymer in the polymer-rich phase, and hydrophobicity of the polymer-rich phase (by for instance adding a hydrophobic organic solvent which partitions to the polymer rich phase) [Alred et al., 1993]. The difference in hydrophobicity between the phases in an EOPO/water system is large and hydrophobic

Figure 7. Partitioning of oligopeptides in Ucon/water two-phase systems. System composition: 20% (w/w) polymer 80% (w/w) water, 100 mM NaClO4: Concentrations: amino acids 1-10 mM, oligopeptides 1 mg/mL 10 mM Na-phosphate buffer, pH 6.0. Temperature: 60 °C (data from Johansson et al., 1997b).

cosolutes are easily separated from hydrophilic cosolutes. The system has been used to separate amino acids, oligopeptides, and polypeptides [Johansson et al., 1995, 1997b]. The partition coefficient of amino acids in Ucon/water two-phase systems has been studied [Johansson et al., 1995]. The partition coefficients of the different amino acids reflect the hydrophobicities of the side groups, where tryptophan is the most hydrophobic amino acid. Hydrophilic amino acids such as lysine and glycine are strongly excluded from the polymer-rich phase. The Ucon/water two-phase system can therefore be used to purify tryptophan from other amino acids or other hydrophilic cosolutes. Oligopeptides have also been separated according to differences in hydrophobicity. For homopeptides the preference for the water phase or the polymer-rich phase becomes more pronounced with increasing degree of polymerization of the peptide (Figure 7) [Johansson et al., 1997b]. The mixing entropy in the polymer-rich phase is lower than the mixing entropy in the water-rich phase. This difference in mixing entropy between the phases corresponds to a driving force which tends to drive the cosolutes to partition to the water phase. One could say that the polymer-rich phase exerts an “entropic repulsion” against all cosolutes [Johansson et al., 1998]. If the partitioning is performed at a higher temperature the polymer-rich phase becomes more concentrated with polymer which leads to an increased entropic repulsion. This effect can be exemplified by tryptophan, which has a K value of 0.64 at 60 °C and 3.3 at 100 °C in a thermoseparated Ucon/water two-phase system [Johansson et al., 1997b]. However, if the cosolute is sufficiently hydrophobic, the partitioning of the cosolute to the polymer rich phase may increase upon temperature increase. This effect has been observed for polytryptophan in the Ucon/water two-phase system [Johansson et al., 1997b]. The partitioning of a charged cosolute to the EOPOrich phase is facilitated by counterions which have relatively low hydrophilicity. These effects are exemplified in Figure 8 where the partitioning of positively charged tryptophan (pH 2) to the polymer rich phase was enhanced when its counterion was exchanged from SO42- to ClO4- [Johansson et al., 1995]. Large monovalent ions such as (C2H5)3NH+, I-, SCNand ClO4- have relatively low hydrophilicity and has been used as counterions for target proteins to enhance the partitioning of the target protein to the EOPO copolymer-rich top phase in two-polymer aqueous two-

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Figure 8. Partitioning of tryptophan in a Ucon/water two-phase system. Effect of pH and different anions on the partitioning. Salt type: Na2SO4 (O), NaCl (0), NaClO4 (]). System composition: 20% (w/w) polymer, 80% (w/w) water, 100 mM salt (50 mM in the case of Na2SO4) and 2 mM Trp. Temperature 60 °C (data from Johansson et al., 1995).

phase systems [Berggren et al., 1995; Johansson et al., 1996]. The same strategy has been used in the onepolymer thermoseparated two-phase system, where the partitioning of positively and negatively charged tryptophan was strongly enhanced to the polymer-rich phase with the addition of ionic surfactants such as sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB), respectively [Johansson et al., 1995]. A positively charged amphiphilic polypeptide composed of randomly distributed lysinyl and tryptophanyl residues could be quantitatively transferred to the polymerrich phase or the water phase with the addition of NaClO4 or Na2SO4, respectively [Johansson et al., 1997b]. The hydrophobically modified EOPO polymer, HMEOPO, has a low cloud point and a water/HM-EOPO system could therefore be obtained at room temperature [Johansson et al., 1999]. Both phases contain a high concentration of water, >90% (Figure 2). This system should be cost-efficient and suitably for large-scale use, since it contains only low concentrations of one polymer. The water/HM-EOPO system is also an efficient system for Apo A-1M purification. Thus, the cell-free E. coli extract containing Apo A-1M was partitioned in the water/HM-EOPO system. Most contaminating proteins partition into the water phase, and in this way a significant purification factor is achieved. In general, all water-soluble protein will partition into the water phase in a water/HM-EOPO system if there is no direct interaction between the polymer and the protein. Apo A-1M can be back-extracted from the HM-EOPO phase into a water phase by altering the pH, salt composition, and temperature. In this way Apo A-1M was recovered in a water phase and the HM-EOPO polymer was recycled into a new extraction system. In these experiments, where 4 mg/mL of protein was added, the purification factor of Apo A-1M was 7-fold, (a maximum of 10-fold purification could be obtained), and the overall yield after back-extracting the protein from the HMEOPO phase to a new water phase was 90% [Johansson et al., 1999]. Conclusions We have reviewed our recent experimental work on biomolecule purification with temperature-induced phase separation. The use of thermoseparating polymers in aqueous two-phase systems has great advantages as the polymer can be recycled, and the protein can be recov-

ered in a water phase. Analysis has shown that 8590% of the polymer can be recycled after thermoseparation. When using EOPO copolymers no proteins are partitioned to the copolymer phase after thermoseparation. The partitioning of a target protein to the EOPO phase can be enhanced by addition of affinity ligand. The ligand can be covalently linked to the thermoseparating polymer and the polymer-ligand can be recovered in the EOPO phase after thermoseparation. Nonionic surfactants can be added to improve purification of amphiphilic proteins. The surfactants can be recycled together with EOPO copolymer after thermal separation. It is also possible to use two thermoseparating polymers to create an aqueous two-phase system. In this case either phase can be used for extraction of the target protein since both phases can be thermoseparated. Small peptides can be separated in a water/EOPO two-phase system. Hydrophobic peptides will partition to the copolymer phase and the hydrophilic peptides will partition to the water phase. Salt effects can be used to influence the partitioning. In water/HM-EOPO systems, it is possible also to partition larger molecules, e.g., proteins, between the two phases because of the high water content in the copolymer-enriched phase. The use of thermoseparating polymers in aqueous two-phase extraction has improved the performance of this separation method. This type of system is attractive for primary recovery of biomolecules where the target molecule can be rapidly extracted to a thermoseparating polymer phase. The recycling of copolymer is important for development of environmentally favorable downstream processes. Acknowledgment This work was supported by grants from the Swedish Center for Bioseparation and the Swedish Research Council for Engineering Sciences (TFR). Literature Cited Albertsson, P.-A° ; Partitioning of cell particles and macromolecules, 3rd ed.; Wiley: New York, 1986. Alred, P. A.; Kozlowski, A.; Harris, J. M.; Tjerneld, F. Application of temperature-induced phase partitioning at ambient temperature for enzyme purification. J. Chromatogr. 1994, 659, 289298. Alred, P. A.; Modlin, R. F.; Tjerneld, F. Partitioning of ecdysteroids using temperature-induced phase separation. J. Chromatogr. 1993, 628, 205-214. Alred, P. A.; Tjerneld, F.; Kozlowski, A.; Harris, J. M. Synthesis of dye conjugates of ethylene oxide-propylene oxide copolymers and application in temperature-induced phase partitioning. Bioseparation 1992, 2, 363-373. Ananthapadmanabhan, K. P.; Goddard, E. D. The relationship between clouding and aqueous biphase formation in polymer solution. Colloid. Surf. 1987, 25, 393. Bailey, F. E.; Callard, R. W. Some properties of poly(ethylene oxide) in aqueous solution. J. Appl. Polym. Sci. 1959, 1, 5662. Berggren, K.; Johansson, H.-O.; Tjerneld, F. Effects of salts and the surface hydrophobicity of proteins on partitioning in aqueous two-phase systems containing thermoseparating ethylene oxide-propylene oxide copolymers. J. Chromatogr. A 1995, 718, 67-79. Bradford, M. M. A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248-254. Carlsson, A.; Karlstro¨m, G.; Lindman, B.; Stenberg, O. Interaction between ethyl(hydroxyethyl)cellulose and sodium dodecyl sulphate in aqueous solution. Colloid Polym. Sci. 1988, 266, 10311036.

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Ind. Eng. Chem. Res., Vol. 39, No. 8, 2000

Creighton, T. Proteins structure and molecular properties, 2nd edition; W. H. Freeman and Company: New York, 1993. Galaev, I.; Mattiasson, B. Thermoreactive water-soluble polymers, nonionic surfactants, and hydrogels as reagents in biotechnology. Enzyme Microb. Technol. 1993, 15, 354-366. Harris, P. A.; Karlstro¨m, G.; Tjerneld, F. Enzyme purification using temperature-induced phase formation. Bioseparation 1991, 2, 237-246. Hudson, C. S. Die gegenseitige lo¨slichkeit von nikotin in wasser. Phys. Chem. 1904, 47, 113-115. Hustedt, H.; Kroner, K. H.; Kula, M.-R. In Partitioning in aqueous two-phase systems. Theory, method, uses and applications to biotechnology; Walter, H., Brooks, D. E., Fisher, D., Eds.; Academic Press: New York, 1985; pp 529-587. Johansson, H.-O.; Karlstro¨m, G.; Tjerneld, F. Experimental and theoretical study of phase separation in aqueous solutions of clouding polymers and carboxylic acids. Macromolecules 1993, 26, 4478-4483. Johansson, H.-O.; Karlstro¨m, G.; Mattiasson, B.; Tjerneld, F. Effects of hydrophobicity and counterions on the partitioning of amino acids in thermoseparating Ucon-water two-phase systems. Bioseparation 1995, 5, 269-279. Johansson, H.-O.; Lundh, G.; Karlstro¨m, G.; Tjerneld, F. Effects of ions on partitioning of serum albumin and lysozyme in aqueous two-phase systems containing ethylene oxide/propylene oxide copolymers. Biochim. Biophys. Acta 1996, 1290, 290-298. Johansson, H.-O.; Karlstro¨m, G.; Tjerneld, F. Effect of solute hydrophobicity on phase behaviour in solutions of thermoseparating polymers. Colloid Polym. Sci. 1997a, 275, 458-466. Johansson, H.-O.; Karlstro¨m, G.; Tjerneld, F. Temperatureinduced phase partitioning of peptides in water solutions of ethylene oxide and propylene oxide random copolymer. Biochim. Biophys. Acta 1997b, 1335, 315-325. Johansson, H.-O.; Karlstro¨m, G.; Tjerneld, F.; Haynes, C. A. Driving forces for phase separation and partitioning in aqueous two-phase systems. J. Chromatogr. 1998, 711, 3-17. Johansson, H.-O.; Persson, J.; Tjerneld, F. Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification. Biotechnol. Bioeng. 1999, 66, 247-257. Louai, A.; Sarazin, D.; Pollet, G.; Franc¸ ois, J.; Moreaux, F. Properties of ethylene oxide-propylene oxide statistical copolymers in aqueous solution. Polymer 1991a, 32, 703-712. Louai, A.; Sarazin, D.; Pollet, G.; Franc¸ ois, J.; Moreaux, F. Effect of additives on solution properties of ethylene oxide-propylene oxide statistical copolymer. Polymer 1991b, 32, 713-720.

Persson, J.; Nystro¨m, L.; Ageland, H.; Tjerneld, F. Purification of recombinant apolipoprotein A-1Milano expressed in E. coli using aqueous two-phase extraction followed by temperature induced phase separation. J. Chromatogr. 1998, 711, 97-109. Persson, J.; Nystro¨m, L.; Ageland, H.; Tjerneld, F. Purification of recombinant and human apolipoprotein A-1 using surfactant micelles in aqueous two-phase systems; recycling of thermoseparating polymer and surfactant with temperature induced phase separation. Biotechnol. Bioeng. 1999a, 65, 371-381. Persson, J.; Johansson, H.-O.; Tjerneld, F. Purification of protein and recycling of polymers in a new aqueous two-phase system using two thermoseparating polymers. J. Chromatogr. A 1999b, 864, 31-48. Rogers, R. D.; Bond, A. H.; Bauer, C. B.; Zhang, J.; Griffin, S. T. Metal ion separation in poly(ethylene glycol)-based aqueous biphasic systems: correlation of partitioning behavior with available thermodynamic hydration data. J. Chromatogr. B 1996, 680, 221-229. Saeki, S.; Kuwahara, N.; Nakata, M.; Kaneko, M. Upper and lower critical solution temperature in poly (ethylene glycol) solutions. Polymer 1976, 17, 685-689. Shinoda, K. Principles of solution and solubility; Marcel Dekker Inc., New York, 1978. Sjo¨berg, A° ; Karlstro¨m, G.; Tjerneld, F. Temperature dependence of the phase equilibria for the system poly (ethylene glycol)/ dextran/water. A theoretical and experimental study. Macromolecules 1989, 22, 4512-4516. Tjerneld, F.; Berner, S.; Cajarville, A.; Johansson, G. New aqueous two-phase system based on hydroxypropyl starch useful in enzyme purification. Enzyme Microb. Technol. 1986, 8, 417423. Walter, H., Johansson, G., Eds. Aqueous two-phase systems. Methods in Enzymology; Academic Press: New York, 1994; Vol. 228 Walter, H.; Brooks, D. E.; Fisher, D. Partitioning in aqueous twophase systems; Academic Press: Orlando, FL, 1985. Woker, R.; Vernau, J.; Kula, M.-R. In Purification of oxinitrilases from plants. Methods in enzymology; Walter, H., Johansson, G., Eds.; Academic Press: London, 1994; Vol. 228, pp 584-590.

Received for review June 25, 1998 Revised manuscript received April 3, 2000 Accepted April 23, 2000 IE9804125