Canadian Journal on Chemical Engineering & Technology Vol. 2, No.

2, February 2011

Advances in Aqueous Two-Phase Systems and Applications in Protein Separation and Purification
Y. Liu, Y.L. Yu, M.Z. Chen and X. Xiao
Department of Biology, College of Science, Shantou University, Shantou 515063, People's Republic of China liuyanglft@stu.edu.cn
Abstract Aqueous two-phase systems are utilized or considered for large-scale downstream processing of proteins extraction using a few stages. This review has briefly summarized the application of aqueous two-phase systems for protein separation and purification, highlighted various aqueous two-phase systems design composed of polymer/polymer type, polymer/salt type, surfactant type, and ionic liquid type. It illustrates especially the potential application of the surfactant-based aqueous two-phase system consisting of single surfactant, mixed surfactant, or surfactant/polymer, respectively. In short, it is pointed out that it is of importance to develop recycle and universal aqueous two-phase system for protein separation and purification in the downstream processing of biotechnology. Key Words aqueous two-phase system, polymer, salt, surfactant, ionic liquids, protein separation

I. INTRODUCTION Conventionally, an aqueous two-phase system (ATPS) will form when two types of water soluble polymers or a water soluble polymer and a low molecular weight substance (inorganic salt in general) dissolve in aqueous solution above their critical concentrations. The top phase is rich in one polymer, and the bottom phase is rich in the other polymer or the salt. The two phases both contain water at high proportion (about 80~99% by weight), and possess extremely low interfacial tensions (on the order of 10-5Nm-1). Albertsson developed the extensive ATPSs and their components originally[1]. Each ATPS with different components can be characterized by its unique phase diagram, which contains the equilibrium phase compositions for one phase turning into two phases of this system. ATPSs provide different physical and chemical environments which allow for the partitioning of biomolecules such as proteins[2,3], plasmid DNA[4-6], toxin[7] and trypsin[8,9] and so on. These biomolecules partitioning in ATPS is influenced by many variables such as salt type and concentration, pH, phase forming polymer type, molecular weight and concentration, temperature, and the structural properties of the biomolecules. The small differences in the top and the bottom phases avoid harsh treatment to extractive comparing with traditional organic solvent/aqueous extraction systems, which provide a protective environment for biomolecules with wide molecular dimension[10,11]. 1

Two development directions of ATPS gradually emerge with ATPS practical applications. First, it is reduction of the components cost and development novel cheap components to comprise ATPS. Second, it is to increase the components reused performance and development the recycle components. Up to now, there are four main types of ATPS: (1) polymer/polymer[12], (2) polymer/salt[13], (3) surfactant[14], and (4) ionic liquid/salt[15]. For the last two types, they have just developed into novel ATPSs for these years. This paper reviews the recent development of the various ATPSs for protein separation and purification which composed of the four types of components above. Especially, it introduces the new components of the ATPSs other than the traditional polymer/polymer such as polyethylene glycol (PEG)/dextran or polymer/salt such as polyethylene glycol/phosphates. Moreover, it discusses the characteristic properties of the novel ATPSs consisted of surfactant or ionic liquid. Finally, it is summarized the universal and potential applications of the ATPSs in downstream processing of biotechnology. II. AQUEOUS TWO-PHASE SYSTEM OF POLYMER/POLYMER A. Polymer Type Comprising Aqueous Two-Phase System To ATPS of polymer/polymer, polymer type, molecular weight and concentration have a great effect on protein partitioning in ATPS. Polyethylene glycol (PEG) and dextran with high molecular weight are the two polymers frequently used to form ATPSs due to their stable physical or chemical structure, and non-toxic characteristic for biological materials. However, there are mainly three problems of PEG/dextran ATPS in practical application. Firstly, the dextran-rich phase in a PEG/dextran ATPS has high viscosity (102~103 mPas) increasing with raising concentration of dextran, which bring the difficult of phase separation. Secondly, dextran is too expensive to scale-up extraction process. The last point to be emphasized is that there have difficulties to carry out cyclic utilization of polymers. To solve above problems, many novel polymers to comprise ATPS have emerged these years. In search of polymers alternative of ATPS, scientists have tried to explore the possibility of substituting this expensive dextran with low-cost polymers such as plasma substitute (cheaper dextran)[16], xanthan[17], and starch derivatives[18] et al. Another kind of substitutes are recyclable polymers of ATPS, which are listed in Table 1. As shown in Table 1, there are high polymer recycle rates (about above 80%) of the

Canadian Journal on Chemical Engineering & Technology Vol. 2, No. 2, February 2011 recyclable polymers. The solubility of these polymers forming ATPS is controlled by various physical conditions (temperature, pressure, pH, and light). Therefore, the polymers can be recycled through exchanging operation conditions after extraction process. It should be noted that the ATPS of EOPO[18] or HM-EOPO[20] in second and third rows of Table 1 is comprised by single polymer. These polymers separated from the water solution over their critical temperature (the cloud point) and a new ATPS is formed where a water phase is equilibrium with a polymer phase with millecular-like structures. So the top phase of these ATPSs contains 100% (w/w) water and the bottom phase contains polymer with a certain concentration. The cloud point of the polymer is not too high (above 40 ) for the separation of biological materials, which cause a few polymers (mostly synthesized) can form APTS applying in biotechnology. ligands to enhance the partition of target molecules. The PEG molecule has become the further target of modification with a wide range of ligand molecules because most proteins partition preferentially to the dextran-rich phase and PEG is easily amenable to derivatization due to its terminal hydroxyl groups[29].
TABLE APPLICATIONS OF AFFINITY ATPS Affinity Target protein molecules PEG IgG[29] diglutaric acid GFPC10G1 CBM9 d[30] Choline GFP-C-lytA[31]

ATPS PEG/Dextran C10G1c PEG/Dextran PEG/Phosphate PEG/Dextran

Yield (%)a 96 -70 83 --

Kb -3.1 ---

TABLE I COMPARISON OF THE RECYCLABLE POLYMERS OF ATPS Polymer Type of Polymer molecules recycle rate Extractive polymer (%) a EOPO L-asparaginase[19] 81.3-84.7 ThermoHM-EOPOb Apolipoprotein -sensitive NIPAM-VI/HMA-1[20] 53/92 EOPOc BSA[21] Pressure-Amino acide[22] PEG/NH4NH2CO4 sensitive pHLysozyme, PNBC/PADBd 98/97 sensitive BSA[12] LightPNNC/PADBe 98/97 BSA, L-Tyr[23] sensitive a EOPO: ethylene oxidepropylene oxide polymers. b HM-EOPO: a hydrophobically modified random polymer of EO and PO with aliphatic C14H29- groups coupled to the end of the polymer. c NIPAM-VI: copolymers of 1-vinylimidazole (VI) with Nisopropylacrylamide (NIPAM). d PNBC: copolymer synthesized by using n-isopropylacrylamide, n-Butyl acrylate, chlorophyllin sodium copper salt as monomers; PADB: copolymer synthesized by using acrylic acid, 2-(dimethylamino) ethyl methacrylate, and n-butyl methacrylate as monomers, and ammonium persulfate and sodium hydrogen sulfite as initiators. e PNNC: copolymer synthesized by using N-isopropylacrylamide, N-vinyl2-pyrrolidone, chlorophyllin sodium copper salt as monomers, and 2,2-azobisisobutyronitrile as initiator.

Cu(II)IDA>10 His6-GFP[27] PEG PEG/Dextran PEG-COOH IgG[32] 93 -PEG/Dextran Human IgG90.8 -f IgG [25] PEG/Phosphate HRPe 78.2 -a Yield: target protein extraction yield. b K: the target protein partition coefficient is defined as a ratio of the top concentration to the bottom concentration. c C10G1: the nonionic surfactant n-decyl -D-glucopyranoside. d CBM9: a family 9 carbohydrate-binding module. e HRP: horseradish peroxidase. f IgG: rabbit anti-human IgG.

Over the years, the advent of genetic engineering and molecular biology create new opportunities and challenges for those seeking to gain efficient and low-cost separation and purification methods. Affinity ATPS is high selectivities, relatively rapid, cost-effective and suitable for large-scale preparations. Table 2 lists some applications about affinity ATPS which have been reported recently. As shown in Table 2, there are two main protein products of green fluorescent protein (GFP) and immunoglobulin G (IgG) which are the topic biologic agents widely applied as biological indicators and therapies medicines respectively. Various affinity interaction can mostly cause a high extraction yield of the target protein. Therefore, affinity ATPS would potentially ensure low downstream costs suiting to the expected upstream of genetic engineering or molecular biology gain. III. AQUEOUS TWO-PHASE SYSTEM OF POLYMER/SALT ATPS of polymer/salt is relatively cheap to ATPS of polymer/polymer (only 1/3-1/5 cost of polymer/polymer), so this kind of ATPS has wide application in biotechnology. Of the polymer/salt systems uses, PEG-phosphate is particularly preferred due to important advantages, including extensive characterization, low cost, low viscosity (101~102 mPas), rapid phase splitting and a wide range of applications[33]. The practical ATPS separation strategies can be divided into four main stages: (i) initial physicochemical characterization of the target proteins solution; (ii) selection of the type of ATPS (types of polymers or salts forming ATPS); (iii) selection of system parameters (molecular weight of the polymer, tie-line 2

B. Affinity Aqueous Two-Phase System Affinity relies on specific, reversible and non-covalent interactions between two biological molecules that form a complex[24]. Affinity ligands specific for particular target proteins can be used to increase selectivity and purification factor of target product in ATPS separation, and the ligands can be free or attached covalently to one of the phase-forming components[25]. In general, there are two fundamental modalities of affinity ligands. One is specific ligands such as substrates, inhibitors or antibodies with defined affinity for single proteins[26], the other is group ligand such as triazine dye and metal chelating ligand with broad affinity for the common structure characteristic proteins[27,28].Both PEG and dextran molecules have been modified with different type of

Canadian Journal on Chemical Engineering & Technology Vol. 2, No. 2, February 2011 length, volume ratio and pH); and (iv) evaluation of the influence of process parameters (sample load, neutral salts addition, consecutive extraction stages et al) upon product recovery/purity[34]. For traditional PEG-phosphate ATPS, the partition behavior of the target products and contaminants under different system parameters is experimentally evaluated usually as a first step in separation process, so optimal conditions under which the product and the contaminants concentrate in opposite phases are selected through lots of experiments. A properly designed systematic approach is required in order to reduce this large number of experiments[35]. Selection of key parameters is an important task and requires thorough knowledge of solute to be studied. In view of this, experiments are planned through a systematic approach[36], wherein all the parameters that are expected to affect the partitioning of target proteins are considered in a logical sequence. In general, the target protein of fermentation broth is enriched in polymer phase (top phase) while the cell debris and contaminants are enriched in salt phase (bottom phase). Therefore, to separate the target protein from the polymer solution becomes an essential process as ATPS separation subsequently. In theory, adding appropriate salt into the polymer phase to form a new ATPS, the target protein can be back-extracted and concentrated in to salt phase and most of the polymer can be recovered. Unfortunately, practical situation is not ideal for the target protein or the polymer, which causes separation of target protein from polymer phase to be the major bottlenecks of ATPS. There are some attempts to separate target protein by common unit operations such as ultrafiltration[37] and chromatograph[38]. Nevertheless, these operations combined with ATPS would increase the cost of isolation target protein. How to improve characteristic of recyclable ATPS still is the research interests for most of biotechnology researchers. III. SURFACTANT-BASED AQUEOUS TWO-PHASE SYSTEM A. Aqueous Two-Phase System Based on Single Surfactant Single surfactant (mostly nonionic surfactant) solution has also been used to form ATPS for protein extraction and separation upon heating above its cloud point[39], which is similar to the ATPS comprising by single thermo-sensitive polymer mentioned earlier. Figure 1 shows a common phase diagram of an ATPS composed with single surfactant. It reveals that the two phases (M point) consist of a surfactantdepleted phase (P1 point) and a surfactant-rich phase (P2 point). When surfactant concentration reaches above the critical micellar concentration (CMC), the surfactant molecules can form spontaneously micellar structure in aqueous because they are amphipathic and easy to assemble above their CMC. There are more and larger micelles in surfactant-rich phase, while fewer and smaller micelles in surfactant-depleted phase. So the two phases also are named 3 micelle-rich and micelle-poor phases. The micelles of the ATPS provide hydrophobic, closed and independent space for biological materials, which can avoid biomolecules denaturation due to aggregating[40]. Therefore, the ATPS based on single surfactant is suitable to extract and separate hydrophobic biomolecules such as membrane proteins in surfactant-rich phase, while hydrophilic contaminants are extracted in surfactant-depleted phase[41].
36 34 surfactant (w/w%) temperature ( )
temperature ( )

30
28 26 24 22 20
P1

32

biphasic part

P2

homogeneous phase part

0 5 surfactant 10 15 20 (w/w%) Fig. 1. Phase diagram of an aqueous two-phase system composed with single surfactant.

Few surfactants can form ATPS about room temperature that is fit for biomolecules separation due to different cloud points of surfactants. To solve this problem, many works about phase equilibria in ATPS based on surfactants have been done recently[42]. It is indicated that various salt addition into ATPS of single surfactant can change the cloud points of surfactants[43], which brings extended surfactant applying in ATPS for biomolecules extraction and separation. Up to now, the nonionic surfactants such as polyoxyethylene alkyl ethers and polyoxyethylene t-octyl-p-phenyl ether are usually used to form ATPS. To control temperature and salt concentration in ATPS separation, the surfactant can be recycled in a great measure[44]. B. Aqueous Two-Phase System Based on Mixed Surfactants More and more ATPSs based on mixed surfactants (cationic surfactant and anion surfactant) have been used for biomolecules separation with development of mixed surfactants phase diagrams. There have ever been some difficulties for mixed surfactants forming ATPS because cation surfactant and anion surfactant are prone to precipitation in aqueous solution by electrostatic interaction[45]. It indicates that the pseudo ternary phase diagram of cationic surfactant and anionic surfactant mixtures at low total surfactant concentrations contains two narrow twophase regions, which represent two kinds of different ATPS systems formed when cationic and anionic surfactants are in excess, respectively (called ATPS-C and ATPS-A). Figure 2 shows a common phase diagram of the ATPS composed with mixed surfactant. The phase separation is similar to ATPS based on single surfactant above, one phase is surfactant-rich, and the other phase is surfactant-depleted[46]. Unlike single surfactant ATPS, mixed surfactants ATPS has more complex

Canadian Journal on Chemical Engineering & Technology Vol. 2, No. 2, February 2011 microstructure not only including spherical micelles but also including rod-shaped micelles, vesicles and lamellar phase due to the different molecular constitution of mixed surfactants[45]. There are two main advantages of mixed surfactants ATPS for biomolecules separation on the basis of its microstructure. Firstly, the high water content (up to 99%) in both top and bottom phases can be provided in favor of biological materials due to the low total surfactant concentrations. Secondly, positive or negative charges in ATPS based on cationic and anionic surfactants can bring strong electrostatic interaction with biomolecules in extraction process, which is controlled by adding counter ions of different salts. However, the two-phase regions comprised by mixed surfactants are usually too narrow to be applied in biomolecules separation. Many investigations have been paid on extending the two-phase regions, such as adding some short chain fatty alcohol (ethanol, n-propanol and n-butanol) or inorganic salt (NaF, Na2SO4 and Na3PO4), increasing temperature and adjusting pH value[47-49]. The cationic surfactant or anionic surfactant can be precipitated and recycled through varying the two-phase region.
H2O

solubility than polymer/polymer ATPS, and has better hydrophilic protein solubility and stability than single surfactant ATPS. Table 3 summarizes the surfactant-based ATPSs with different components. As shown in the second column of Table 3, there are lower concentrations (about 10-1~10-2 mol/L) of the total surfactants to form the surfactant-based ATPS. They are also named as aqueous micellar two-phase systems (AMTPSs) because most of surfactant-based ATPSs comprise micelle-rich structure in the top phase as listed in the third column of Table 3. It should be noted that the mixed surfactants ATPS can extract proteins with higher concentration (reached to 1 g/L) than other types of AMTPS perhaps due to the complex microstructure of mixed surfactant ATPS. As shown in the sixth column of Table 3, ATPS comprised of surfactant and polymer can better serve extract hydrophobic membrane protein with the high partition coefficient reaching to 60.
TABLE COMPARISON OF SURFACTANT-BASED ATPSS Surfactant/total MicelleExtractive/ Type concentration rich ConcentraVt/Vb Ka (mol/L) phase tion (g/L) Lysozyme[53] Top Single C10E4b/0.12 4.0 0.78 /0.10 c Top 0.33 27e surfactant C12EO5 /0.12 d COX [54] /-f C10E4-C10TAB G6PDg [55] Mixed /0.128 Top -7.7 /0.68 Top 0.58 20.8 surfactants C10NEBSA[47]/1.0 C10SO3h/0.07 COX[52]/0.3i Bacteriorhod60 opsin[52]/0.3i Surfactant/ C12EO511 Top -polymer PEG40000/0.11 BSA[52]/0.5i 0.30 0.59 Lysozyme[52] /0.5i a K: the target protein partition coefficient is defined as a ratio of the top concentration to the bottom concentration. b C10E4: the nonionic surfactant n-decyl tetra(ethylene oxide). c C12EO5: the nonionic surfactant penta ethylene glycol mono-n-dodecyl ether. d COX: cholesterol oxidase. e This K value is caculated by the yield (90%) and the volume ratio (1/3) in original text. f C10TAB: the cationic surfactants n-decyltrimethylammonium bromide. g G6PD: glucose-6-phosphate dehydrogenase. h C10NE-C10SO3: the cationic surfactants decyltriethylammonium bromide and the anionic surfactant sodium decylsulfonate, respectively. i This group protein concentration unit is mg/g.

ATPS-C ATPS-A

homogeneous phase part

homogeneous phase part

anion surfactant

cation surfactant

Fig. 2. Phase diagram of an aqueous two-phase system composed with mixed surfactants.

C. Aqueous Two-Phase System of Surfactant/Polymer A range of nonionic surfactants above their CMC can form ATPS with water-soluble polymers such as PEG or dextran without temperature increasing as the single surfactant ATPS. This kind of surfactant/polymer ATPS is successfully utilized to extract selectively a variety of integral membrane proteins from cytoplasmic and peripheral membrane proteins for purification and characterization. Much work about ATPS of surfactant/polymer has been done by Tjernelds group[50-52]. In the ATPS of surfactant/polymer, the surfactant micelles can be regarded as the second polymer of the ATPS whose phase diagram is similar to that of polymer/polymer ATPS. Surfactant/polymer ATPS can increase the partitioning of hydrophilic proteins to the polymer phase and enrich membrane proteins into the surfactant phase. Therefore, the surfactant/polymer ATPS has better membrane proteins 4

V. IONIC LIQUID-BASED AQUEOUS TWO-PHASE SYSTEM Ionic liquids (ILs) are usually composed of large organic cations and smaller inorganic anions. They are roomtemperature liquid salts with neglectable vapor pressure and tunable structure, which facilitates ILs applications in catalysis, extraction and material application. ILs-based aqueous two-phase extraction is a novel ATPS extraction in biological separation. Rogers and coworkers have firstly reported the phase diagrams of ILs-based ATPS[56]. Kragl et al. have investigated the potential of a special class of ILs for

Canadian Journal on Chemical Engineering & Technology Vol. 2, No. 2, February 2011 the formation of biocompatible ATPS[57]. These ILs are applicable for the formation of ATPS when the inorganic salt KH2PO4/K2HPO4 (10-25%, w/w) is used as the second phase forming compound. Above critical concentrations of these two compounds in aqueous solution, phase separation takes place resulting in the formation of an IL-enriched upper and a saltenriched bottom phase. It is indicated that the IL-based ATPS offers the opportunity to combine the purification process of active biocatalysts with the performance of enzyme-catalyzed reactions especially in case of the conversion of hydrophobic substrates. Subsequently, ATPS based on ILs have been utilized in various biomaterials separation and purification. Lius group has developed an ATPS based on [BMIM][Cl] that was applied to extract opium alkaloids for a sample pretreatment prior to HPLC[58]. The same IL has been also used for the extraction of testosterone and epitestosterone from human urine by ATPS with extraction efficiencies of 8090% for both analytes[59]. The extraction of proteins by ILs-based ATPS has been first realized by Du et al. who extracted proteins from human body fluids by employing a [C4MIM][Cl]/K2HPO4 system[60]. It is indicated that hydrophobic interactions, electrostatic interactions and salting-out effects are important for the transfer of the proteins[15,61]. IX. CONCLUSION Recent developments outlined of ATPS have suggested that there is considerable scope for further innovation, including the examination of new ATPSs and their compositions. The developing understanding of the nature of partition depends primarily on the properties of compositions in ATPS. This review has indicated that various compositions including polymer/polymer, polymer/salt, surfactant, and ionic liquid of aqueous two-phase system are the key factors for ATPS application in protein separation and purification. Moreover, it is evident that the reduced commerical application of ATPSs is caused by the complexity of the construction of the new forming phase compositions and the low process recovery reported. Therefore, the use of low-cost ATPS easy to recycle, the considerable potential to achieve process integration and the increasing application of ATPS for the separation and purification of high-value bioproducts with a few stages is providing a new opportunity for biotechnology downstream areas. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (No. 21006062). REFERENCES
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BIOGRAPHIES

Y. Liu (1978-, born in Meilongjiang Province, China) Education: Ph.D., biochemical engineering, Tianjin University, China. Employments: associate professor, Shantou University, China. Part-time jobs: reviewers of some academic journals such as Biochemical engineering journal and Journal of Chemical Technology & Biotechnology. Research interests: bioseparation, novel extraction technique, surfactant chemistry. Y.L. Yong (1987-, born in Gansu Province, China) Education: B.Sc., Hunan Agricultural University, China. Employments: graduate student, Shantou University, China. Research interests: bioseparation.

M.Z. Chen (1956-, born in Fujian Province, China) Education: M.Sc., Hainan University, China. Employments: professor, Shantou University, China. Research interests: biotechnology.

NSERT
X. Xiao (1978-, born in Guangdong Province, China) Education: B.Sc., Shantou University, China. Employments: senior laboratory technician, Shantou University, China. Research interests: biotechnology.