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PEG–salt aqueous two-phase systems: an attractive and versatile liquid–liquid extraction technology for the downstream processing of proteins and enzymes

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Abstract

Nowadays, there is an increasing demand to establish new feasible, efficient downstream processing (DSP) techniques in biotechnology and related fields. Although several conventional DSP technologies have been widely employed, they are usually expensive and time-consuming and often provide only low recovery yields. Hence, the DSP is one major bottleneck for the commercialization of biological products. In this context, polyethylene glycol (PEG)–salt aqueous two-phase systems (ATPS) represent a promising, efficient liquid–liquid extraction technology for the DSP of various biomolecules, such as proteins and enzymes. Furthermore, ATPS can overcome the limitations of traditional DSP techniques and have gained importance for applications in several fields of biotechnology due to versatile advantages over conventional DSP methods, such as biocompatibility, technical simplicity, and easy scale-up potential. In the present review, various practical applications of PEG–salt ATPS are presented to highlight their feasibility to operate as an attractive and versatile liquid–liquid extraction technology for the DSP of proteins and enzymes, thus facilitating the approach of new researchers to this technique. Thereby, single- and multi-stage extraction, several process integration methods, as well as large-scale extraction and purification of proteins regarding technical aspects, scale-up, recycling of process chemicals, and economic aspects are discussed.

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References

  • Aguilar O, Albiter V, Serrano-Carreón L, Rito-Palomares M (2006) Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli. J Chromatogr B 835(1–2):77–83. doi:10.1016/j.jchromb.2006.03.016

    CAS  Google Scholar 

  • Aguirre C, Concha I, Vergara J, Riveros R, Illanes A (2010) Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous two-phase systems. Process Biochem 45(7):1163–1167. doi:10.1016/j.procbio.2010.03.002

    CAS  Google Scholar 

  • Åkerlund H-E (1984) An apparatus for counter-current distribution in a centrifugal acceleration field. J Biochem Biophys Methods 9(2):133–141. doi:10.1016/0165-022X(84)90004-6

    PubMed  Google Scholar 

  • Albertsson P-Å (1965) Thin-layer countercurrent distribution. Anal Biochem 11(1):121–125. doi:10.1016/0003-2697(65)90050-3

    CAS  PubMed  Google Scholar 

  • Albertsson P-Å (1986) Partition of cell particles and macromolecules. Wiley, New York

    Google Scholar 

  • Albertsson P-Å, Johansson G, Tjerneld F (1990) Aqueous two-phase separations. In: Asenjo JA (ed) Separation processes in biotechnology Bioprocess Technology, vol 9. Marcel Dekker, New York, pp 287–327

    Google Scholar 

  • Alcântara LAP, Minim LA, Minim VPR, Bonomo RCF, da Silva LHM, da Silva MCH (2011) Application of the response surface methodology for optimization of whey protein partitioning in PEG/phosphate aqueous two-phase system. J Chromatogr B 879(21):1881–1885. doi:10.1016/j.jchromb.2011.05.007

    Google Scholar 

  • Ambler CM (1959) The theory of scaling up laboratory data for the sedimentation type centrifuge. J Biochem Microbiol Technol Eng 1(2):185–205. doi:10.1002/jbmte.390010206

    CAS  Google Scholar 

  • Andersson E, Hahn-Hägerdal B (1990) Bioconversions in aqueous two-phase systems. Enzym Microb Technol 12(4):242–254. doi:10.1016/0141-0229(90)90095-8

    CAS  Google Scholar 

  • Andersson E, Mattiasson B, Hahn-Hägerdal B (1984) Enzymatic conversion in aqueous two-phase systems: deacylation of benzylpenicillin to 6-aminopenicillanic acid with penicillin acylase. Enzym Microb Technol 6(7):301–306. doi:10.1016/0141-0229(84)90057-7

    Google Scholar 

  • Andrews BA, Asenjo JA (2010) Theoretical and experimental evaluation of hydrophobicity of proteins to predict their partitioning behavior in aqueous two phase systems: a review. Sep Sci Technol 45(15):2165–2170. doi:10.1080/01496395.2010.507436

    CAS  Google Scholar 

  • Andrews BA, Head DM, Dunthorne P, Asenjo JA (1990) PEG activation and ligand binding for the affinity partitioning of proteins in aqueous two-phase systems. Biotechnol Tech 4(1):49–54. doi:10.1007/BF00156610

    CAS  Google Scholar 

  • Azevedo AM, Gomes AG, Rosa PAJ, Ferreira IF, Pisco AMMO, Aires-Barros MR (2009a) Partitioning of human antibodies in polyethylene glycol–sodium citrate aqueous two-phase systems. Sep Purif Technol 65(1):14–21. doi:10.1016/j.seppur.2007.12.010

    CAS  Google Scholar 

  • Azevedo AM, Rosa PAJ, Ferreira IF, Aires-Barros MR (2009b) Chromatography-free recovery of biopharmaceuticals through aqueous two-phase processing. Trends Biotechnol 27(4):240–247. doi:10.1016/j.tibtech.2009.01.004

    CAS  PubMed  Google Scholar 

  • Azevedo AM, Rosa PAJ, Ferreira IF, Pisco AMMO, de Vries J, Korporaal R, Visser TJ, Aires-Barros MR (2009c) Affinity-enhanced purification of human antibodies by aqueous two-phase extraction. Sep Purif Technol 65(1):31–39. doi:10.1016/j.seppur.2008.03.006

    CAS  Google Scholar 

  • Bach E, Sant’Anna V, Daroit DJ, Corrêa APF, Segalin J, Brandelli A (2012) Production, one-step purification, and characterization of a keratinolytic protease from Serratia marcescens P3. Process Biochem 47(12):2455–2462. doi:10.1016/j.procbio.2012.10.007

    CAS  Google Scholar 

  • Banik RM, Santhiagu A, Kanari B, Sabarinath C, Upadhyay SN (2003) Technological aspects of extractive fermentation using aqueous two-phase systems. World J Microbiol Biotechnol 19(4):337–348. doi:10.1023/A:1023940809095

    CAS  Google Scholar 

  • Barbosa JMP, Souza RL, Fricks AT, Zanin GM, Soares CMF, Lima ÁS (2011) Purification of lipase produced by a new source of Bacillus in submerged fermentation using an aqueous two-phase system. J Chromatogr B 879(32):3853–3858. doi:10.1016/j.jchromb.2011.10.035

    CAS  Google Scholar 

  • Belter PA, Cussler EL, Hu W-S (1988) Bioseparations. Wiley, New York

    Google Scholar 

  • Benavides J, Rito-Palomares M (2006) Simplified two-stage method to B-phycoerythrin recovery from Porphyridium cruentum. J Chromatogr B 844(1):39–44. doi:10.1016/j.jchromb.2006.06.029

    CAS  Google Scholar 

  • Benavides J, Rito-Palomares M (2008) Practical experiences from the development of aqueous two-phase processes for the recovery of high value biological products. J Chem Technol Biotechnol 83(2):133–142. doi:10.1002/jctb.1844

    CAS  Google Scholar 

  • Benavides J, Aguilar O, Lapizco-Encinas BH, Rito-Palomares M (2008) Extraction and purification of bioproducts and nanoparticles using aqueous two-phase systems strategies. Chem Eng Technol 31(6):838–845. doi:10.1002/ceat.200800068

    CAS  Google Scholar 

  • Benavides J, Rito-Palomares M, Asenjo JA (2011) Aqueous two-phase systems. In: Moo-Young M (ed) Comprehensive biotechnology. Principles and practices in industry, agriculture, medicine and the environment, 2nd edn. Academic Press, Burlington, pp 697–713

    Google Scholar 

  • Bezerra RP, Borba FKSL, Moreira KA, Lima-Filho JL, Porto ALF, Chaves AC (2006) Extraction of amylase from fermentation broth in poly (ethylene glycol) salt aqueous two-phase system. Braz Arch Biol Technol 49(4):547–555. doi:10.1590/S1516-89132006000500003

    CAS  Google Scholar 

  • Bhavsar K, Ravi Kumar V, Khire JM (2012) Downstream processing of extracellular phytase from Aspergillus niger: chromatography process vs. aqueous two phase extraction for its simultaneous partitioning and purification. Process Biochem 47(7):1066–1072. doi:10.1016/j.procbio.2012.03.012

    CAS  Google Scholar 

  • Bim MA, Franco TT (2000) Extraction in aqueous two-phase systems of alkaline xylanase produced by Bacillus pumilus and its application in kraft pulp bleaching. J Chromatogr B 743(1–2):349–356. doi:10.1016/S0378-4347(00)00223-1

    CAS  Google Scholar 

  • Birkenmeier G, Vijayalakshmi MA, Stigbrand T, Kopperschläger G (1991) Immobilized metal ion affinity partitioning, a method combining metal-protein interaction and partitioning of proteins in aqueous two-phase systems. J Chromatogr A 539(2):267–277. doi:10.1016/S0021-9673(01)83935-2

    CAS  Google Scholar 

  • Boychyn M, Doyle W, Bulmer M, More J, Hoare M (2000) Laboratory scaledown of protein purification processes involving fractional precipitation and centrifugal recovery. Biotechnol Bioeng 69(1):1–10. doi:10.1002/(SICI)1097-0290(20000705)69:1, <1::AID-BIT1>3.0.CO;2-4

    CAS  PubMed  Google Scholar 

  • Bradoo S, Saxena RK, Gupta R (1999) Partitioning and resolution of mixture of two lipases from Bacillus stearothermophilus SB-1 in aqueous two-phase system. Process Biochem 35(1–2):57–62. doi:10.1016/S0032-9592(99)00032-1

    CAS  Google Scholar 

  • Cabral JMS (2007) Cell partitioning in aqueous two-phase polymer systems. In: Kumar A, Galaev I, Mattiasson B (eds) Cell separation, vol 106, Advances in biochemical engineering/biotechnology. Springer, Berlin, pp 151–171

    Google Scholar 

  • Canales M, Ballesteros C, Moreno-Cid JA, Espinosa AM, Villar M, de la Fuente J (2009) Extractive bioconversion to produce the Aedes albopictus akirin in an aqueous two-phase system supporting Pichia pastoris growth and protein secretion. Biochem Eng J 46(2):105–114. doi:10.1016/j.bej.2009.04.014

    CAS  Google Scholar 

  • Chang HN, Lee Y-H, Lee CY (1992) Continuous production of 6-APA in an aqueous two-phase system. Ann NY Acad Sci 672(1):643–648. doi:10.1111/j.1749-6632.1992.tb35684.x

    CAS  PubMed  Google Scholar 

  • Chen J-P, Wang C-H (1991) Lactose hydrolysis by β-galactosidase in aqueous two-phase systems. J Ferment Bioeng 71(3):168–175. doi:10.1016/0922-338X(91)90104-O

    CAS  Google Scholar 

  • Chouyyok W, Wongmongkol N, Siwarungson N, Prichanont S (2005) Extraction of alkaline protease using an aqueous two-phase system from cell free Bacillus subtilis TISTR 25 fermentation broth. Process Biochem 40(11):3514–3518. doi:10.1016/j.procbio.2005.03.052

    CAS  Google Scholar 

  • Cordes A, Kula M-R (1986) Process design for large-scale purification of formate dehydrogenase from Candida boidinii by affinity partition. J Chromatogr B 376:375–384. doi:10.1016/S0378-4347(00)80853-1

    CAS  Google Scholar 

  • Cordes A, Flossdorf J, Kula MR (1987) Affinity partitioning: development of mathematical model describing behavior of biomolecules in aqueous two-phase systems. Biotechnol Bioeng 30(4):514–520. doi:10.1002/bit.260300408

    CAS  PubMed  Google Scholar 

  • Costa M, Cunha M, Cabral JS, Aires-Barros M (2000) Scale-up of recombinant cutinase recovery by whole broth extraction with PEG-phosphate aqueous two-phase. Bioseparation 9(4):231–238. doi:10.1023/A:1008162209596

    CAS  PubMed  Google Scholar 

  • Cunha T, Aires-Barros R (2002) Large-scale extraction of proteins. Mol Biotechnol 20(1):29–40. doi:10.1385/MB:20:1:029

    CAS  PubMed  Google Scholar 

  • Cunha MT, Costa MJL, Calado CRC, Fonseca LP, Aires-Barros MR, Cabral JMS (2003) Integration of production and aqueous two-phase systems extraction of extracellular Fusarium solani pisi cutinase fusion proteins. J Biotechnol 100(1):55–64. doi:10.1016/S0168-1656(02)00225-0

    CAS  PubMed  Google Scholar 

  • Datar R (1986) Economics of primary separation steps in relation to fermentation and genetic engineering. Process Biochem 21(1):19–26

    CAS  Google Scholar 

  • de Gouveia T, Kilikian BV (2000) Bioaffinity extraction of glucoamylase in aqueous two-phase systems using starch as free bioligand. J Chromatogr B 743(1–2):241–246. doi:10.1016/S0378-4347(00)00031-1

    Google Scholar 

  • Diamond AD, Hsu JT (1992) Aqueous two-phase systems for biomolecule separation. In: Fiechter A (ed) Bioseparation. Advances in biochemical engineering, vol 47. Springer, Berlin, pp 89–135

    Google Scholar 

  • Dismer F, Alexander Oelmeier S, Hubbuch J (2013) Molecular dynamics simulations of aqueous two-phase systems: understanding phase formation and protein partitioning. Chem Eng Sci 96:142–151. doi:10.1016/j.ces.2013.03.020

    CAS  Google Scholar 

  • Dos Reis CJ, Thömmes J, Kula M-R (1994) Continuous separation of whey proteins with aqueous two-phase systems in a Graesser contactor. J Chromatogr A 668(1):85–94. doi:10.1016/0021-9673(94)80095-2

    Google Scholar 

  • Dos Reis CJ, Thömmes J, Meirelles AJ, Kula MR (1995) Performance of a Graesser contactor in the continuous extraction of whey proteins: mixing, mass transfer and efficiency. Bioseparation 5(5):259–268

    Google Scholar 

  • Espitia-Saloma E, Vázquez-Villegas P, Aguilar O, Rito-Palomares M (2014) Continuous aqueous two-phase systems devices for the recovery of biological products. Food Bioprod Process 92(2):101–112. doi:10.1016/j.fbp.2013.05.006

    CAS  Google Scholar 

  • Fauquex P-F, Hustedt H, Kula M-R (1985) Phase equilibration in agitated vessels during extractive enzyme recovery. J Chem Technol Biotechnol 35(1):51–59. doi:10.1002/jctb.280350110

    Google Scholar 

  • Fernandes S, Johansson G, Hatti-Kaul R (2001) Purification of recombinant cutinase by extraction in an aqueous two-phase system facilitated by a fatty acid substrate. Biotechnol Bioeng 73(6):465–475. doi:10.1002/bit.1081

    CAS  PubMed  Google Scholar 

  • Gavasane MR, Gaikar VG (2003) Aqueous two-phase affinity partitioning of penicillin acylase from E. coli in presence of PEG-derivatives. Enzym Microb Technol 32(6):665–675. doi:10.1016/S0141-0229(03)00032-2

    CAS  Google Scholar 

  • Greve A, Kula M-R (1991) Recycling of salts in partition protein extraction processes. J Chem Technol Biotechnol 50(1):27–42. doi:10.1002/jctb.280500105

    CAS  PubMed  Google Scholar 

  • Gu T (2000) Liquid-liquid partitioning methods for bioseparations. In: Satinder A (ed) Separation science and technology, vol 2, handbook of bioseparations. Academic Press, New York, pp 329–364

    Google Scholar 

  • Guan Y, Wu X-Y, Treffry TE, Lilley TH (1992) Studies on the isolation of penicillin acylase from Escherichia coli by aqueous two-phase partitioning. Biotechnol Bioeng 40(4):517–524. doi:10.1002/bit.260400410

    CAS  PubMed  Google Scholar 

  • Guan Y, Lilley TH, Treffry TE, Zhou C-L, Wilkinson PB (1996) Use of aqueous two-phase systems in the purification of human interferon-α1 from recombinant Escherichia coli. Enzym Microb Technol 19(6):446–455. doi:10.1016/S0141-0229(96)00051-8

    CAS  Google Scholar 

  • Harris JM (1985) Labratory synthesis of polyethylene glycol derivatives. J Macromol Sci, Part C: Polym Rev J 25(3):325–373. doi:10.1080/07366578508081960

    Google Scholar 

  • Harris JM, Struck EC, Case MG, Paley MS, Yalpani M, Van Alstine JM, Brooks DE (1984) Synthesis and characterization of poly(ethylene glycol) derivatives. J Polym Sci: Polym Chem Ed 22(2):341–352. doi:10.1002/pol.1984.170220207

    CAS  Google Scholar 

  • Hart RA, Lester PM, Reifsnyder DH, Ogez JR, Builder SE (1994) Large scale, in situ isolation of periplasmic IGF-I from E. coli. Nat Biotechnol 12(11):1113–1117. doi:10.1038/nbt1194-1113

    CAS  Google Scholar 

  • Head DM, Andrews BA, Asenjo JA (1989) Epoxy-oxirane activation of PEG for protein ligand coupling. Biotechnol Tech 3(1):27–32. doi:10.1007/BF01876217

    CAS  Google Scholar 

  • Hotha S, Banik RM (1997) Production of alkaline protease by Bacillus thuringiensis H 14 in aqueous two-phase systems. J Chem Technol Biotechnol 69(1):5–10. doi:10.1002/(SICI)1097-4660(199705)69:1, <5::AID-JCTB661>3.0.CO;2-I

    CAS  Google Scholar 

  • Hummel W, Schütte H, Kula M-R (1983) Large scale production of D-lactate dehydrogenase for the stereospecific reduction of pyruvate and phenylpyruvate. Eur J Appl Microbiol Biotechnol 18(2):75–85. doi:10.1007/BF00500828

    CAS  Google Scholar 

  • Hummel W, Schütte H, Kula M-R (1985) D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei. Appl Microbiol Biotechnol 21(1-2):7–15. doi:10.1007/BF00252354

    CAS  Google Scholar 

  • Hustedt H (1986) Extractive enzyme recovery with simple recycling of phase forming chemicals. Biotechnol Lett 8(11):791–796. doi:10.1007/BF01020824

    CAS  Google Scholar 

  • Hustedt H, Papamichael N (1988) Automated continuous crosscurrent extraction of proteins. Ann NY Acad Sci 542(1):135–139. doi:10.1111/j.1749-6632.1988.tb25818.x

    CAS  PubMed  Google Scholar 

  • Hustedt H, Kroner K-H, Menge U, Kula M-R (1980) Enzyme purification by liquid-liquid extraction. In: Weetall HH, Royer GP (eds) Enzyme engineering, vol 5. Springer, New York, pp 45–47

    Google Scholar 

  • Hustedt H, Kroner K-H, Kula M-R (1985a) Applications of phase partitioning in biotechnology. In: Walter H, Brooks DE, Fisher D (eds) Partitioning in aqueous two-phase system theory, methods, uses, and applications to biotechnology, vol 1. Academic Press, London, pp 529–587

    Google Scholar 

  • Hustedt H, Kroner K-H, Menge U, Kula M-R (1985b) Protein recovery using two-phase systems. Trends Biotechnol 3(6):139–144. doi:10.1016/0167-7799(85)90102-7

    CAS  Google Scholar 

  • Hustedt H, Börner B, Kroner KH, Papamichael N (1987) Fully automated continuous crosscurrent extraction of enzymes in a two-stage plant. Biotechnol Tech 1(1):49–54. doi:10.1007/BF00156287

    CAS  Google Scholar 

  • Hustedt H, Kronor K-H, Papamichael N (1988) Continuous cross-current aqueous two-phase extraction of enzymes from biomass: automated recovery in production scale. Process Biochem 23(5):129–137

    CAS  Google Scholar 

  • Igarashi L, Kieckbusch TG, Franco TT (2004) Xylanase mass transfer studies in aqueous two-phase systems using spray and sieve plate columns. Bioprocess Biosyst Eng 26(3):151–157. doi:10.1007/s00449-003-0329-x

    CAS  PubMed  Google Scholar 

  • Ito Y, Bowman RL (1971) Countercurrent chromatography. Anal Chem 43(13):69A–75A. doi:10.1021/ac60307a767

    CAS  Google Scholar 

  • Jafarabad KR, Sawant SB, Joshi JB, Sikdar SK (1992) Enzyme and protein mass transfer coefficient in aqueous two-phase systems—I. Spray extraction columns. Chem Eng Sci 47(1):57–68. doi:10.1016/0009-2509(92)80200-V

    CAS  Google Scholar 

  • Johansson G (1994) Recovery of proteins and phase-forming chemicals. In: Walter H, Johansson G (eds) Aqueous two-phase systems: methods in enzymology, vol 228, Aqueous two-phase systems. Academic Press, London, pp 569–573

    Google Scholar 

  • Kammoun R, Chouayekh H, Abid H, Naili B, Bejar S (2009) Purification of CBS 819.72 α-amylase by aqueous two-phase systems: modelling using response surface methodology. Biochem Eng J 46(3):306–312. doi:10.1016/j.bej.2009.06.003

    CAS  Google Scholar 

  • Kim CY, Brewer JW, Brothers CE, Farver TF, Lee EK (1988) Recovery of extracellular enzyme from fermentation broth by aqueous phase system. In: 3rd Chemical Congress of North America, Toronto, Canada, June 5-10

  • Kondo A, Urabe T, Higashitani K (1994) Bioconversions in an aqueous two-phase system using enzymes immobilized on ultrafine silica particles. J Ferment Bioeng 77(6):700–703. doi:10.1016/0922-338X(94)90158-9

    CAS  Google Scholar 

  • Kroner K-H, Schütte H, Stach W, Kula M-R (1982) Scale-up of formate dehydrogenase by partition. J Chem Technol Biotechnol 32(1):130–137. doi:10.1002/jctb.5030320117

    CAS  Google Scholar 

  • Kroner KH, Hustedt H, Kula M-R (1984) Extractive enzyme recovery: economic considerations. Process Biochem 19:170–179

    Google Scholar 

  • Kuhlmann W, Halwachs W, Schügerl K (1980) Racemat-Spaltung von Aminosäuren durch Immobilisierung von Acylase im wäßrigen Zweiphasensystem. Chem Ing Tech 52(7):607–607. doi:10.1002/cite.330520720

    CAS  Google Scholar 

  • Kula M-R, Selber K (2002) Protein purification, aqueous liquid extraction. In: Flickinger MC, Drew SW (eds) Encyclopedia of bioprocess technology, vol 4, Fermentation, biocatalysis, and bioseparation. Wiley, New York, pp 2179–2191

    Google Scholar 

  • Kula M-R, Kroner K-H, Hustedt H (1982) Purification of enzymes by liquid-liquid extraction. In: Fiechter A (ed) Reaction engineering. Advances in biochemical engineering, vol 24. Springer, Berlin, pp 73–118

    Google Scholar 

  • Liao L-C, Ho CS, Wu W-T (1999) Bioconversion with whole cell penicillin acylase in aqueous two-phase systems. Process Biochem 34(5):417–420. doi:10.1016/S0032-9592(98)00099-5

    CAS  Google Scholar 

  • Ling Y-Q, Nie H-L, Su S-N, Branford-White C, Zhu L-M (2010) Optimization of affinity partitioning conditions of papain in aqueous two-phase system using response surface methodology. Sep Purif Technol 73(3):343–348. doi:10.1016/j.seppur.2010.04.020

    CAS  Google Scholar 

  • Liu Y, Yu YL, Chen MZ, Xiao X (2011) Advances in aqueous two-phase systems and applications in protein separation and purification. Can J Chem Eng Technol 2(2):1–7

    Google Scholar 

  • Madhusudhan MC, Raghavarao KSMS (2011) Aqueous two phase extraction of invertase from baker’s yeast: effect of process parameters on partitioning. Process Biochem 46(10):2014–2020. doi:10.1016/j.procbio.2011.07.014

    CAS  Google Scholar 

  • Madhusudhan MC, Raghavarao KSMS, Nene S (2008) Integrated process for extraction and purification of alcohol dehydrogenase from Baker’s yeast involving precipitation and aqueous two phase extraction. Biochem Eng J 38(3):414–420. doi:10.1016/j.bej.2007.08.007

    CAS  Google Scholar 

  • Mannweiler K (1989) The recovery of biological particles in high-speed continuous centrifuges with special reference to feed-zone break-up effects. Dissertation, University of London

  • Mao LN, Rogers JK, Westoby M, Conley L, Pieracci J (2010) Downstream antibody purification using aqueous two-phase extraction. Biotechnol Prog 26(6):1662–1670. doi:10.1002/btpr.477

    CAS  PubMed  Google Scholar 

  • Marcos JC, Fonseca LP, Ramalho MT, Cabral JMS (1998) Variation of penicillin acylase partition coefficient with phase volume ratio in poly(ethylene glycol)–sodium citrate aqueous two-phase systems. J Chromatogr B 711(1–2):295-299. 10.1016/S0378-4347(97)00633-6

  • Marini A, Imelio N, Picó G, Romanini D, Farruggia B (2011) Isolation of a Aspergillus niger lipase from a solid culture medium with aqueous two-phase systems. J Chromatogr B 879(22):2135–2141. doi:10.1016/j.jchromb.2011.05.042

    CAS  Google Scholar 

  • Mayolo-Deloisa K, González-Valdez J, Guajardo-Flores D, Aguilar O, Benavides J, Rito-Palomares M (2011) Current advances in the non-chromatographic fractionation and characterization of PEGylated proteins. J Chem Technol Biotechnol 86(1):18–25. doi:10.1002/jctb.2498

    CAS  Google Scholar 

  • Minami NM, Kilikian BV (1998) Separation and purification of glucoamylase in aqueous two-phase systems by a two-step extraction. J Chromatogr B 711(1–2):309–312. doi:10.1016/S0378-4347(98)00039-5

    CAS  Google Scholar 

  • Mohammadi HS, Omidinia E (2013) Process integration for the recovery and purification of recombinant Pseudomonas fluorescens proline dehydrogenase using aqueous two-phase systems. J Chromatogr B 929:11–17. doi:10.1016/j.jchromb.2013.03.024

    CAS  Google Scholar 

  • Muendges J, Stark I, Mohammad S, Górak A, Zeiner T (2015) Single stage aqueous two-phase extraction for monoclonal antibody purification from cell supernatant. Fluid Phase Equilib 385:227–236. doi:10.1016/j.fluid.2014.10.034

    CAS  Google Scholar 

  • Mukataka S, Haynes CA, Prausnitz JM, Blanch HW (1992) Extractive bioconversions in aqueous two-phase systems: enzymatic hydrolysis of casein proteins. Biotechnol Bioeng 40(2):195–206. doi:10.1002/bit.260400202

    CAS  PubMed  Google Scholar 

  • Naganagouda K, Mulimani VH (2008) Aqueous two-phase extraction (ATPE): an attractive and economically viable technology for downstream processing of Aspergillus oryzae α-galactosidase. Process Biochem 43(11):1293–1299. doi:10.1016/j.procbio.2008.07.016

    CAS  Google Scholar 

  • Nascimento KS, Rosa PAJ, Nascimento KS, Cavada BS, Azevedo AM, Aires-Barros MR (2010) Partitioning and recovery of Canavalia brasiliensis lectin by aqueous two-phase systems using design of experiments methodology. Sep Purif Technol 75(1):48–54. doi:10.1016/j.seppur.2010.07.008

    CAS  Google Scholar 

  • Nascimento CO, Soares PAG, Porto TS, Costa RMPB, Lima CA, de Lima Filho JL, Coelho LCBB, dos Santos Correia MT, da Cunha C, MdG PALF (2013) Aqueous two-phase systems: new strategies for separation and purification of lectin from crude extract of Cratylia mollis seeds. Sep Purif Technol 116:154–161. doi:10.1016/j.seppur.2013.05.012

    CAS  Google Scholar 

  • Nfor BK, Ahamed T, van Dedem GWK, van der Wielen LAM, van de Sandt EJAX, Eppink MHM, Ottens M (2008) Design strategies for integrated protein purification processes: challenges, progress and outlook. J Chem Technol Biotechnol 83(2):124–132. doi:10.1002/jctb.1815

  • Ng HS, Tan CP, Chen SK, Mokhtar MN, Ariff A, Ling TC (2011) Primary capture of cyclodextrin glycosyltransferase derived from Bacillus cereus by aqueous two phase system. Sep Purif Technol 81(3):318–324. doi:10.1016/j.seppur.2011.07.039

    CAS  Google Scholar 

  • Nitsawang S, Hatti-Kaul R, Kanasawud P (2006) Purification of papain from Carica papaya latex: aqueous two-phase extraction versus two-step salt precipitation. Enzym Microb Technol 39(5):1103–1107. doi:10.1016/j.enzmictec.2006.02.013

    CAS  Google Scholar 

  • Oelmeier SA, Dismer F, Hubbuch J (2011) Application of an aqueous two-phase systems high-throughput screening method to evaluate mAb HCP separation. Biotechnol Bioeng 108(1):69–81. doi:10.1002/bit.22900

    CAS  PubMed  Google Scholar 

  • Omidinia E, Shahbaz Mohamadi H, Dinarvand R, Taherkhani H-A (2010) Investigation of chromatography and polymer/salt aqueous two-phase processes for downstream processing development of recombinant phenylalanine dehydrogenase. Bioprocess Biosyst Eng 33(3):317–329. doi:10.1007/s00449-009-0327-8

    CAS  PubMed  Google Scholar 

  • Papamichael N, Hustedt H (1994) Enzyme recovery by continuous crosscurrent extraction. In: Walter H, Johansson G (eds) Aqueous two-phase systems, vol 228. Academic Press, San Diego, pp 573–584

    Google Scholar 

  • Papamichael N, Börner B, Hustedt H (1992) Continuous aqueous phase extraction of proteins: Automated processing and recycling of process chemicals. J Chem Technol Biotechnol 54(1):47–55. doi:10.1002/jctb.280540110

    CAS  Google Scholar 

  • Park H-M, Lee S-W, Chang W-J, Koo Y-M (2007) Affinity separation by protein conjugated IgG in aqueous two-phase systems using horseradish peroxidase as a ligand carrier. J Chromatogr B 856(1–2):108–112. doi:10.1016/j.jchromb.2007.05.036

    CAS  Google Scholar 

  • Pavlou AK, Reichert JM (2004) Recombinant protein therapeutics—success rates, market trends and values to 2010. Nat Biotechnol 22(12):1513–1519. doi:10.1038/nbt1204-1513

    CAS  PubMed  Google Scholar 

  • Platis D, Labrou NE (2006) Development of an aqueous two-phase partitioning system for fractionating therapeutic proteins from tobacco extract. J Chromatogr A 1128(1–2):114–124. doi:10.1016/j.chroma.2006.06.047

    CAS  PubMed  Google Scholar 

  • Porto TS, Silva GM M e, Porto CS, Cavalcanti MTH, Neto BB, Lima-Filho JL, Converti A, Porto ALF, Pessoa A Jr (2008) Liquid–liquid extraction of proteases from fermented broth by PEG/citrate aqueous two-phase system. Chem Eng Process 47(4):716–721. doi:10.1016/j.cep.2006.12.004

    CAS  Google Scholar 

  • Prinz A, Koch K, Górak A, Zeiner T (2014) Multi-stage laccase extraction and separation using aqueous two-phase systems: experiment and model. Process Biochem 49(6):1020–1031. doi:10.1016/j.procbio.2014.03.011

    CAS  Google Scholar 

  • Raghavarao KSMS, Rastogi NK, Gowthaman MK, Karanth NG (1995) Aqueous two-phase extraction for downstream processing of enzymes/proteins. In: Saul LN, Allen IL (eds) Advances in applied microbiology, vol 41. Academic Press, New York, pp 97–171

    Google Scholar 

  • Raja S, Murty VR, Thivaharan V, Rajasekar V, Ramesh V (2011) Aqueous two phase systems for the recovery of biomolecules—a review. Sci Technol 1(1):7–16. doi:10.5923/j.scit.20110101.02

    Google Scholar 

  • Ratanapongleka K (2010) Recovery of biological products in aqueous two phase systems. Int J Chem Eng Appl 1(2):191–198. doi:10.7763/IJCEA.2010.V1.33

    CAS  Google Scholar 

  • Rito-Palomares M (2004) Practical application of aqueous two-phase partition to process development for the recovery of biological products. J Chromatogr B 807(1):3–11. doi:10.1016/j.jchromb.2004.01.008

    CAS  Google Scholar 

  • Rito-Palomares M, Hernandez M (1998) Influence of system and process parameters on partitioning of cheese whey proteins in aqueous two-phase systems. J Chromatogr B 711(1–2):81–90. doi:10.1016/S0378-4347(98)00011-5

    CAS  Google Scholar 

  • Rito-Palomares M, Lyddiatt A (1996) Impact of cell disruption and polymer recycling upon aqueous two-phase processes for protein recovery. J Chromatogr B 680(1–2):81–89. doi:10.1016/0378-4347(95)00500-5

    CAS  Google Scholar 

  • Rito-Palomares M, Lyddiatt A (2000) Practical implementation of aqueous two-phase processes for protein recovery from yeast. J Chem Technol Biotechnol 75(7):632–638. doi:10.1002/1097-4660(200007)75:7, <632::AID-JCTB248>3.0.CO;2-7

    CAS  Google Scholar 

  • Rito-Palomares M, Lyddiatt A (2002) Process integration using aqueous two-phase partition for the recovery of intracellular proteins. Chem Eng J 87(3):313–319. doi:10.1016/S1385-8947(01)00241-8

    CAS  Google Scholar 

  • Rito-Palomares M, Middelberg APJ (2002) Aqueous two-phase systems for the recovery of a recombinant viral coat protein from Escherichia coli. J Chem Technol Biotechnol 77(9):1025–1029. doi:10.1002/jctb.673

    CAS  Google Scholar 

  • Rito-Palomares M, Huddleston JG, Lyddiatt A (1994) Phase recycling in aqueous two-phase partition processes: impact upon practical implementation of protein recovery from brewery waste. In: Pyle DL (ed) Separations for biotechnology 3, vol 158. Royal Society of Chemistry, Cambridge, pp 413–419

    Google Scholar 

  • Rito-Palomares M, Dale C, Lyddiatt A (2000) Generic application of an aqueous two-phase process for protein recovery from animal blood. Process Biochem 35(7):665–673

    CAS  Google Scholar 

  • Rito-Palomares M, Nuñez L, Amador D (2001) Practical application of aqueous two-phase systems for the development of a prototype process for c-phycocyanin recovery from Spirulina maxima. J Chem Technol Biotechnol 76(12):1273–1280. doi:10.1002/jctb.507

    CAS  Google Scholar 

  • Rosa PAJ, Azevedo AM, Sommerfeld S, Mutter M, Aires-Barros MR, Bäcker W (2009) Application of aqueous two-phase systems to antibody purification: a multi-stage approach. J Biotechnol 139(4):306–313. doi:10.1016/j.jbiotec.2009.01.001

    CAS  PubMed  Google Scholar 

  • Rosa PAJ, Azevedo AM, Sommerfeld S, Bäcker W, Aires-Barros MR (2011) Aqueous two-phase extraction as a platform in the biomanufacturing industry: economical and environmental sustainability. Biotechnol Adv 29(6):559–567. doi:10.1016/j.biotechadv.2011.03.006

    CAS  PubMed  Google Scholar 

  • Ruiz-Ruiz F, Benavides J, Aguilar O, Rito-Palomares M (2012) Aqueous two-phase affinity partitioning systems: current applications and trends. J Chromatogr A 1244:1–13. doi:10.1016/j.chroma.2012.04.077

    CAS  PubMed  Google Scholar 

  • Ruiz-Ruiz F, Benavides J, Rito-Palomares M (2013) Scaling-up of a B-phycoerythrin production and purification bioprocess involving aqueous two-phase systems: practical experiences. Process Biochem 48(4):738–745. doi:10.1016/j.procbio.2013.02.010

    CAS  Google Scholar 

  • Schütte H, Kroner KH, Hummel W, Kula MR (1983) Recent developments in separation and purification of biomolecules. Ann NY Acad Sci 413(1):270–282. doi:10.1111/j.1749-6632.1983.tb47899.x

    PubMed  Google Scholar 

  • Schütte H, Hummel W, Tsai H, Kula M-R (1985) L-leucine dehydrogenase from Bacillus cereus. Appl Microbiol Biotechnol 22(5):306–317. doi:10.1007/BF00582413

    Google Scholar 

  • Sebastião MJ, Cabral JMS, Aires-Barros MR (1996) Improved purification protocol of a Fusarium solani pisi recombinant cutinase by phase partitioning in aqueous two-phase systems of polyethylene glycol and phosphate. Enzym Microb Technol 18(4):251–260. doi:10.1016/0141-0229(95)00045-3

    Google Scholar 

  • Selber K, Tjerneld F, Collén A, Hyytiä T, Nakari-Setälä T, Bailey M, Fagerström R, Kan J, van der Laan J, Penttilä M, Kula M-R (2004) Large-scale separation and production of engineered proteins, designed for facilitated recovery in detergent-based aqueous two-phase extraction systems. Process Biochem 39(7):889–896. doi:10.1016/S0032-9592(03)00198-5

    CAS  Google Scholar 

  • Selvakumar P, Ling TC, Covington AD, Lyddiatt A (2012) Enzymatic hydrolysis of bovine hide and recovery of collagen hydrolysate in aqueous two-phase systems. Sep Purif Technol 89:282–287. doi:10.1016/j.seppur.2012.01.046

    CAS  Google Scholar 

  • Shahriari S, Vossoughi M, Taghikhani V, Safekordi AA, Alemzadeh I (2010) Experimental study and mathematical modeling of partitioning of β-amylase and amyloglucosidase in PEG–salt aqueous two-phase systems. J Chem Eng Data 55(11):4968–4975. doi:10.1021/je100587j

    CAS  Google Scholar 

  • Shibusawa Y, Yamaguchi M, Ito Y (1998) Polyethylene glycol-potassium phosphate aqueous two-phase systems for countercurrent chromatography of proteins. J Liq Chromatogr Relate Technol 21(1-2):121–133. doi:10.1080/10826079808001941

    CAS  Google Scholar 

  • Silva ME, Franco TT (1999) Purification of microbial β-galactosidase from Kluyveromyces fragilis by bioaffinity partitioning. Rev Microbiol 30(4):324–331. doi:10.1590/S0001-37141999000400006

    Google Scholar 

  • Simental-Martínez J, Rito-Palomares M, Benavides J (2014) Potential application of aqueous two-phase systems and three-phase partitioning for the recovery of superoxide dismutase from a clarified homogenate of Kluyveromyces marxianus. Biotechnol Prog 30(6):1326–1334. doi:10.1002/btpr.1979

    PubMed  Google Scholar 

  • Sinha J, Dey PK, Panda T (2000) Aqueous two-phase: the system of choice for extractive fermentation. Appl Microbiol Biotechnol 54(4):476–486. doi:10.1007/s002530000342

    CAS  PubMed  Google Scholar 

  • Strandberg L, Köhler K, Enfors S-O (1991) Large-scale fermentation and purification of a recombinant protein from Escherichia coli. Process Biochem 26(4):225–234. doi:10.1016/0032-9592(91)85004-8

    CAS  Google Scholar 

  • Tanuja S, Srinivas ND, Raghava Rao KSMS, Gowthaman MK (1997) Aqueous two-phase extraction for downstream processing of amyloglucosidase. Process Biochem 32(8):635–641. doi:10.1016/S0032-9592(97)00009-5

    CAS  Google Scholar 

  • Targovnik AM, Cascone O, Miranda MV (2012) Extractive purification of recombinant peroxidase isozyme c from insect larvae in aqueous two-phase systems. Sep Purif Technol 98:199–205. doi:10.1016/j.seppur.2012.08.004

    CAS  Google Scholar 

  • Teotia S, Gupta MN (2001) Reversibly soluble macroaffinity ligand in aqueous two-phase separation of enzymes. J Chromatogr A 923(1–2):275–280. doi:10.1016/S0021-9673(01)00968-2

    CAS  PubMed  Google Scholar 

  • Teotia S, Mondal K, Gupta MN (2006) Integration of affinity precipitation with partitioning methods for bioseparation of chitin binding lectins. Food Bioprod Process 84(1):37–43. doi:10.1205/fbp.05133

    CAS  Google Scholar 

  • Tomáška M, Stred’anský M, Tomašková A, Šturdík E (1995) Lactose hydrolysis in aqueous two-phase system by whole-cell β-galactosidase of Kluyveromyces marxianus. Bioprocess Eng 12(1-2):17–20. doi:10.1007/BF01112988

    Google Scholar 

  • van Winssen FA, Merz J, Czerwonka LM, Schembecker G, Dortmund TU (2014a) Application of the tunable aqueous polymer-phase impregnated resins-technology for protein purification. Sep Purif Technol 136:123–129. doi:10.1016/j.seppur.2014.08.030

    Google Scholar 

  • van Winssen FA, Merz J, Schembecker G (2014b) Tunable aqueous polymer-phase impregnated resins-technology—a novel approach to aqueous two-phase extraction. J Chromatogr A 1329:38–44. doi:10.1016/j.chroma.2014.01.001

    PubMed  Google Scholar 

  • Vázquez-Villegas P, Aguilar O, Rito-Palomares M (2011) Study of biomolecules partition coefficients on a novel continuous separator using polymer-salt aqueous two-phase systems. Sep Purif Technol 78(1):69–75. doi:10.1016/j.seppur.2011.01.023

    Google Scholar 

  • Vázquez-Villegas P, Aguilar O, Rito-Palomares M (2015) Continuous enzyme aqueous two-phase extraction using a novel tubular mixer-settler in multi-step counter-current arrangement. Sep Purif Technol 141:263–268. doi:10.1016/j.seppur.2014.12.005

    Google Scholar 

  • Veide A, Smeds A-L, Enfors S-O (1983) A process for large-scale isolation of β-galactosidase from E. coli in an aqueous two-phase system. Biotechnol Bioeng 25(7):1789–1800. doi:10.1002/bit.260250709

    CAS  PubMed  Google Scholar 

  • Veide A, Lindbäck T, Enfors S-O (1984) Continuous extraction of β-D-galactosidase from Escherichia coli in an aqueous two-phase system: effects of biomass concentration on partitioning and mass transfer. Enzym Microb Technol 6(7):325–330. doi:10.1016/0141-0229(84)90062-0

    CAS  Google Scholar 

  • Walker SG, Lyddiatt A (1998) Aqueous two-phase systems as an alternative process route for the fractionation of small inclusion bodies. J Chromatogr B 711(1–2):185–194. doi:10.1016/S0378-4347(97)00604-X

    CAS  Google Scholar 

  • Walter H, Brooks DE, Fisher D (1985) Partitioning in aqueous two-phase systems: theory, methods, uses, and applications to biotechnology. Academic Press, Orlando

    Google Scholar 

  • Wei D-Z, Zhu J-H, Cao X-J (2002) Enzymatic synthesis of cephalexin in aqueous two-phase systems. Biochem Eng J 11(2–3):95–99. doi:10.1016/S1369-703X(02)00032-3

    CAS  Google Scholar 

  • Yang L, Huo D, Hou C, He K, Lv F, Fa H, Luo X (2010) Purification of plant-esterase in PEG1000/NaH2PO4 aqueous two-phase system by a two-step extraction. Process Biochem 45(10):1664–1671. doi:10.1016/j.procbio.2010.06.018

    CAS  Google Scholar 

  • Yue H, Yuan Q, Wang W (2007) Purification of phenylalanine ammonia-lyase in PEG1000/Na2SO4 aqueous two-phase system by a two-step extraction. Biochem Eng J 37(3):231–237. doi:10.1016/j.bej.2007.05.002

    CAS  Google Scholar 

  • Zijlstra GM, de Gooijer CD, Tramper J (1998) Extractive bioconversions in aqueous two-phase systems. Curr Opin Biotechnol 9(2):171–176. doi:10.1016/S0958-1669(98)80111-0

    CAS  PubMed  Google Scholar 

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Glyk, A., Scheper, T. & Beutel, S. PEG–salt aqueous two-phase systems: an attractive and versatile liquid–liquid extraction technology for the downstream processing of proteins and enzymes. Appl Microbiol Biotechnol 99, 6599–6616 (2015). https://doi.org/10.1007/s00253-015-6779-7

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