838 Chem. Eng. Technol. 2008, 31, No.

6, 838845

Jorge Benavides1 Review

Oscar Aguilar1
Blanca H. Lapizco-Encinas1
Marco Rito-Palomares1
1
Departamento de
Biotecnologa e Ingeniera de
Alimentos, Centro de
Biotecnologa, Tecnolgico de
Monterrey, Mexico.
Extraction and Purification of Bioproducts
and Nanoparticles using Aqueous Two-Phase
Systems Strategies
Aqueous Two-Phase Systems (ATPS) is a primary recovery technique that has
shown great potential for the efficient extraction and purification of high value
biological compounds. The main advantages of this technique include scaling up
feasibility, process integration capability and biocompatibility. In this review, the
efficient use of ATPS for the extraction of proteins, genetic material, low molecu-
lar weight compounds, bioparticles, nanoparticles and cells is highlighted. The
important role of ATPS in process integration, i.e., extractive conversion, extrac-
tive fermentation, cell disruption integrated with product recovery, and extractive
purification, is discussed. A novel approach to protein molecular characterization
combining ATPS and 2-dimension electrophoresis (2-DE) is introduced as a first
step in the process development. Novel approaches for downstream processing
using ATPS and dielectrophoresis are presented. Finally, trends concerning the
application of ATPS strategies to address the future challenges of bioseparation
are discussed.

Keywords: Aqueous Two-Phase Systems, Biological Products, Extraction

Received: February 2, 2008; accepted: March 24, 2008
DOI: 10.1002/ceat.200800068

1 Introduction glycol (PEG)-potassium phosphate, is particularly preferred

Aqueous Two-Phase Systems (ATPS) is a liquid-liquid extrac-
tion technique that has been used to establish bioprocesses for
the primary recovery and partial purification of a variety of
biological products, including proteins, genetic material,
nanoparticles, low molecular weight products, cells and cell or-
ganelles [1, 2]. The main advantages of this technique include
scaling up feasibility, process integration capability and bio-
compatibility. ATPS form when hydrophilic compounds such
as some types of polymers (polyethylene glycol, dextran, poly-
propylene glycol, etc.) and salts (phosphates, sulfates, citrates,
etc.) are combined over certain critical concentrations, result-
ing in the formation of two hydrophilic phases, Fig. 1. There
are three main types of ATPS: (1) polymer-salt, (2) polymer-
polymer, and (3) ATPS constructed with alternative com-
pounds, e.g., ethylene oxide and propylene oxide copolymers
(EOPO), hydroxylpropyl starch (HPS), iminoadiacetic acid
(IAA), etc. [3]. Of the polymer-salt systems uses, polyethylene
Correspondence: Prof. M. Rito-Palomares (mrito@itesm.mx), Depar-
tamento de Biotecnologa e Ingeniera de Alimentos, Centro de
Biotecnologa, Tecnolgico de Monterrey, Ave. Eugenio Garza Sada
2501 Sur, Monterrey, 64849, Mexico.
due to important advantages, including extensive characteriza-
tion, low cost, and a wide range of applications [3]. The first
studies involving ATPS date from the late 1950s and early
1960s, when Albertsson [4] demonstrated the great potential
of this technique for the primary recovery of biological com-
pounds. Furthermore, process integration and intensification
can be achieved using strategies based upon ATPS resulting in
optimized processes that are easy to scale up [13]. Process in-
tegration results when one single unit operation can achieve
the same process objective of two or more discrete processing
stages. Consequently, a reduction of the total number of unit
operations is possible. On the other hand, process intensifica-
tion involves the development of strategies that result in pro-
cess modification to maximize the flow of the biological sus-
pensions that enter the process. Such strategies do not require
an increase in either the total number of stages or size of the
equipment.
Bioprocess development using ATPS is limited by the poor
understanding and characterization of the effect of the system
parameters upon the partitioning of a particular compound.
Commonly, the partition behavior of the target products and
contaminants under different system parameters, e.g., tie-line
length (TLL), phase volume ratio, VR, pH, and sample loading,
etc., is experimentally evaluated as a first step in process devel-

2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.cet-journal.com

Chem. Eng. Technol. 2008, 31, No. 6, 838845 Aqueous Two-Phase Systems 839

Figure 1. Simplified representation of the fractionation of bioparticles in aqueous two-phase processes. Aqueous two-phase systems
(ATPS) are usually applied as primary recovery techniques for major contaminant removal from intra- or extracellular products. ATPS form
when hydrophilic compounds such as some types of polymers (polyethylene glycol, dextran, polypropylene glycol, etc.) and salts (phos-
phates, sulfates, citrates, etc) are combined over certain critical concentrations, resulting in the formation of two hydrophilic phases.

opment. From these studies, optimal conditions under which
the product and the contaminants concentrate in opposite
phases are selected. Although research has been conducted in
this area to facilitate process design and reduce the require-
ment for time-consuming experiments, the true molecular un-
derstanding of solute partitioning is just beginning to be devel-
oped. The establishment of guidelines has allowed the
prediction of the partition behavior of solutes in ATPS based
on their physicochemical properties [1, 5, 6]. Three physico-
chemical properties have been identified to be mainly respon-
sible for governing solute partition behavior in ATPS: (1) mo-
lecular weight, (2) hydrophobicity, and (3) superficial net
electrochemical charge [1, 5]. It is clear that once solute parti-
tioning has been characterized, a process can be designed for
the primary recovery and partial purification of the product of
interest. The practical application of ATPS for the recovery
and purification of bioproducts from diverse expression sys-
tems generates efficient, easily scaled up and bio-compatible
extraction processes [2, 3]. However, the future challenges that
the bioseparation of products faces, e.g., increasing product
concentration will require the consideration of novel ap-
proaches where the use of ATPS based strategies may play an
important role.
This paper focuses on presenting a general overview to high-
light the importance of ATPS for the recovery and partial puri-
fication of biological products. The potential role of ATPS in
process integration is presented. A novel approach using ATPS
coupled to two-dimensional (2D) electrophoresis for the phy-
sicochemical characterization of biomolecules is presented as a
route to facilitate the establishment of bioseparation process
conditions. Dielectrophoresis is introduced as an emerging
technique in the field of bioseparation. Finally, the expected
potential trend of the practical application of ATPS to address
future challenges of bioseparation is discussed.

2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim http://www.cet-journal.com

840 J. Benavides et al.
2 Recovery of Biological Products by ATPS
2.1 Proteins
The use of ATPS for bioprocess development has focused
mainly on proteins, i.e., enzymes, therapeutic proteins includ-
ing antibodies, colored proteins, etc. The aqueous environ-
ment of ATPS confers a considerable advantage of the tech-
nique over other recovery strategies, e.g. the use of solvent.
The use of solvents can negatively affect the structural integrity
of the protein of interest. The biological function of proteins,
as well as most biomolecules, is closely related to its native
structure. Therefore, the conservation of the protein native
structure during downstream stages (recovery and purifica-
tion) is always a major concern.
Several studies have been conducted regarding enzyme pri-
mary recovery and partial purification [13]. The enzymes in-
volved in such studies have diverse applications, including en-
zymes widely used in the food, detergent, paper and
pharmaceutical industries. Aguilar et al. [7] reported the use
of ATPS PEG-potassium phosphate for the recovery and par-
tial purification of penicillin acylase produced by a recombi-
nant strain of E. coli. The authors reported a yield of 97 % and
a purification factor of 3.5 at the top phase when optimum
system parameters (PEG 1450 g/mol, TLL 48.5 % w/w, VR =
1.0, pH = 7 and sample loading 35 % w/w) were used. In
addition, the authors presented a direct cost comparison
between the process developed using ATPS and a homologous
process using chromatography. For this particular case, it
was found that a gross cost reduction of 37 % was achieved
when ATPS were used. This reduction was attributed to the de-
crease of the number of total stages from 7 to 4 and the total
cost of the chemicals involved. Mohamadi and Omidinia [8]
studied the recovery of phenylalanine dehydrogenase produced
by a recombinant strain of Bacillus badius using ATPS PEG-
ammonium sulfate. The authors reported a 95.85 % yield
and a purification factor of 474.3 when optimum system
parameters (PEG 6000 g/mol 8.5 % w/w, ammonium sulfate
17.5 % w/w, NaCl 13 % w/w and pH = 8) were used. The puri-
fication factor obtained in this particular case is remarkably
high, demonstrating the potential of ATPS for the primary re-
covery of biological products and also as a purification tech-
nique.
Extensive research can be found in the areas of primary re-
covery and partial purification of therapeutic proteins using
ATPS. The increasing interest in proteins with medical applica-
tions is reflected in the number of publications related to the
production, recovery and purification of these types of bio-
molecules, particularly antibodies (both monoclonal and poly-
clonal). Rosa et al. [9] reported the use of ATPS polymer-poly-
mer and polymer-salt in the presence of chemically modified
(functionalized) varieties of PEG for the recovery of human
immunoglobulin gamma (IgG) produced by Chinese Hamster
Ovary (CHO) cells. IgG is the most abundant immunoglobu-
lin in serum and has a major role in the proper immune re-
sponse. The authors reported a 93 % yield and a 1.9 purifica-
tion factor when PEG-dextran systems in the presence of
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eng. Technol. 2008, 31, No. 6, 838845
150 g/mol PEG-COOH was used. According to the authors, a
60-fold increase was observed in selectivity when functional-
ized PEG (PEG-COOH) was added to the extraction system.
The addition of chemicals, i.e., modified polymers, metal ions,
polar solvents, detergents, etc., may represent a good strategy
to favor the partition of the product of interest towards a de-
sired phase, increasing the selectivity of the extraction system.
Therefore, the applications of ATPS as a purification technique
can be increased considerably by following this strategy. Stud-
ies concerning the recovery and purification of antibodies
from genetically modified plant expression systems have also
been conducted. Platis and Labrou [10] reported the primary
recovery and partial purification of human anti-human immu-
nodeficiency virus (HIV) monoclonal antibody 2F5 (mAb
2F5) expressed in genetically modified tobacco plants using
ATPS PEG-phosphate. Under selected system parameters
(PEG 1500 g/mol 12 % w/w, phosphate 13 % w/w, and pH = 5)
a 95 % yield and a purification factor of 34, were achieved.
The use of ATPS as a primary recovery technique not only al-
lowed the removal of proteins typically found in tobacco, but
also permitted the removal of phytochemicals such as phenolic
compounds and toxic alkaloids. The authors proved the effi-
ciency of the described extraction system using additional pro-
teins of therapeutic importance, such as neuraminidase
from influenza virus and human anti-HIV monoclonal anti-
body 2G12 (mAb 2G12). The recovery and purification of
PEGylated therapeutic proteins is an increasing area of re-
search [11, 12]. The use of ATPS and related techniques for the
recovery and purification of PEGylated proteins represents an
interesting case study that still needs to be explored.
The recovery and purification of colored biological com-
pounds with application in the food and cosmetic industries
represent another interesting case study, since more people ap-
pear to be concerned about the health problems related to the
use of some synthetic pigments. In addition, some of these
naturally colored compounds have applications in the molecu-
lar technology research area as fluorescent biomarkers. In this
context, the primary recovery and partial purification of phy-
cobiliproteins, an important group of colored proteins found
as accessory pigments in algae and cyanobacteria, represents
an interesting case study. Benavides and Rito-Palomares [13]
reported the primary recovery and partial purification
of B-phycoerythrin (BPE) produced by the microalgae Por-
phyridium cruentum using ATPS PEG-phosphate. The use of
ATPS allowed the integration and intensification of the recov-
ery process by feeding the extraction system with a crude
extract (including cell debris) and using a high sample loading
(40 % w/w). Under selected system parameters (PEG
1000 g/mol 29 % w/w, phosphate 9 % w/w, TLL 45 % w/w, VR
= 4.5 and pH = 7) a yield and a purification factor of 90 % and
4, respectively, were achieved. Alternatively, Bermejo et al. [14]
developed and scaled up a process for the recovery and purifi-
cation of BPE produced by Porphyridium cruentum using
Expanded Bed Adsorption (EBA) chromatography. Under
optimum adsorption conditions a yield close to 80 % was
achieved, i.e., a recovery slightly lower than that obtained
using ATPS.
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Chem. Eng. Technol. 2008, 31, No. 6, 838845
2.2 Genetic Material
The development of new protocols and processes for the re-
covery and purification of DNA and genetic vectors is of great
relevance in the molecular biology field, particularly in appli-
cations related to genetic engineering and genomics. Studies
regarding the use of ATPS for the primary recovery and partial
purification of genetic material, particularly plasmid DNA vec-
tors, have been reported. Trindade et al. [15] reported the use
of ATPS PEG-sulfate as a first stage for the recovery of plasmid
DNA (pDNA) vector. E. coli cell lysate containing pDNA was
directly fed into the extraction system. The influence of the
ATPS parameters upon pDNA was studied and optimum con-
ditions selected. A yield of 100 % at the bottom phase of the
system was achieved when PEG 600 34 % w/w, ammonium sul-
fate 6 % w/w, VR = 9.3 and 20 % w/w lysate loading were used.
The process not only resulted in the complete recovery of
pDNA but also allowed the 3-fold concentration of the prod-
uct of interest. In order to achieve process intensification, the
amount of lysate loaded into the extraction system was in-
creased from 20 to 40 % w/w, resulting in a yield of 85 % and
an 8-fold concentration factor. Duarte et al. [16] reported a
novel approach for the recovery of polyplexes (complexes
formed when pDNA is linked to polymers) using ATPS as the
primary recovery stage. Polyplexes are genetic vectors with po-
tential use in gene therapy. The process developed by the
authors consisted in two consecutive ATPS stages (600 g/mol
PEG-ammonium sulfate and 3350 g/mol PEG-Dextran 110)
followed by ultrafiltration. PEGylated polyethyleneimine
(pPEI) was added in the second ATPS stage as an affinity li-
gand targeted to bind polyplexes. The authors were able to ob-
tain plasmid polyplexes with a 100 % yield after the two ex-
traction stages, while RNA and contaminant proteins were
completely removed. Problems involving the binding of the
polyplexes in the ultrafiltration membrane used for the re-
moval of PEG and dextran caused the yield to reduce signifi-
cantly by 510 %. However, the potential use of affinity ATPS
for the recovery of plasmid polyplexes was demonstrated.
2.3 Bio-Nanoparticles
Research in the field of production, recovery, purification and
application of bio-nanoparticles is attracting the interest of the
scientific community due to the great variety of potential com-
mercial applications of these particles. Common applications
for these particles include delivery vectors for gene therapy,
molecular assemblies for drug delivery and the assembly of
bio-conjugates for the construction of functional nanostruc-
tures. In this context, the recovery and purification of virus-
like particles as delivery vectors for gene therapy represents an
interesting case study. Benavides et al. [17] reported the recov-
ery and partial purification of double layered Rotavirus-Like
Particles (dlRLP) produced by insect cells, i.e., baculovirus ex-
pression system using ATPS PEG-potassium phosphate. dlRLP
have several applications, including vaccination against rota-
virus, potential vectors in gene therapy, construction of func-
tional nanoparticles, etc. The recovery and partial purification
process developed by the authors resulted in an overall yield of
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Aqueous Two-Phase Systems 841
85 %, and a purification factor greater than 50. These results
demonstrated the great potential of ATPS for the recovery of
these particles, particularly when compared with the yields
achieved by commonly used virus-like particle recovery tech-
niques such as cesium chloride and glucose gradients. In addi-
tion, the process developed using ATPS had advantages con-
cerning total processing time, energy consumption and scaling
up capability [17]. An area of increasing interest is the use of
ATPS for the fractionation, assembly and recovery of bio-
nanoparticles. Helfrich et al. [18] have conducted studies em-
ploying gold (Au) and silver (Ag) nanospheres, nanowires and
DNA-derivatized nanowires, regarding the fractionation of
metallic nanoparticles and their bioconjugates. The authors
found that the partition behavior of Au and Ag nanospheres in
ATPS PEG-dextran depends on their size. Small nanospheres
(less than 100 nm in diameter) partitioned between the top
and bottom phases, while larger spheres partitioned at the in-
terface. Concerning the partition behavior of nanowires and
their DNA conjugates, it was found that these nanoparticles
partition at the interface of the system. Furthermore, the
authors demonstrated the binding of Au nanospheres with Au
nanowires via selective DNA hybridization at the ATPS inter-
face, in order to construct functional assemblies. These results
demonstrate the potential of ATPS not only as a technique for
nanoparticle fractionation but also as a technique to carry out
in situ assembly and recovery of bio-nanoparticle assemblies.
2.4 Low Molecular Weight Products
Even though several studies have demonstrated the potential
of ATPS for the fractionation, recovery and partial purification
of high molecular weight biomolecules (such as proteins and
DNA), the number of studies regarding the use of this tech-
nique for low molecular weight products is limited. The ma-
jority of the research in the field of ATPS has focused on pro-
teins and DNA (either genomic or plasmidic) due to the
potential commercial application of the products. The enzy-
matic process revolution, the search for therapeutic proteins,
and the birth and growth of genetic engineering related tech-
niques are factors that have contributed to an increase in the
interest of these types of biomolecules. However, a new in-
creasing interest in the use of low molecular weight com-
pounds for the preventive and treatment of diseases is evident.
In this context, the recovery and partial purification of phyto-
chemicals of low molecular weight with proven nutraceutical
activity represents an interesting case study. Cisneros et al.
[19] reported the recovery of lutein produced by the microalga
Chlorella protothecoides using ATPS PEG-potassium phos-
phate. Lutein is a carotenoid with demonstrated protective ac-
tivity against macular degeneration related with aging. The
authors found that despite the hydrophobic character of lutein,
ATPS showed great potential for its recovery. The addition of
ethanol to the system increased the affinity of lutein for the
polymer phase while no affinity was observed towards the salt-
rich phase. This strategy could be used for the recovery of sim-
ilar hydrophobic compounds. A yield of 81 % was achieved
with selected system parameters (PEG 8000 g/mol 22.9 % w/w,
potassium phosphate 10.3 % w/w, VR = 1, pH = 7 and ethanol
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842 J. Benavides et al. Chem. Eng. Technol. 2008, 31, No. 6, 838845

5 % w/w). Although investigation re-

garding the recovery and purification
of low molecular weight compounds is
still incipient, a boost in this area is
expected in the near future due to the
general interest in nutraceutical com-
pounds.

2.5 Cells and Cell Organelles

Fractionation of whole cells as well as

cell organelles using ATPS has also
been studied. Such studies addressed
different applications including cell
sorting, cellular membrane character-
ization, organelle fractionation and
isolation. Cell sorting on ATPS basi-
cally depends on the physicochemical
properties of the cell membrane. Eda-
hiro et al. [20] reported a selection
method of high-yield cultured straw-
berry cells producing anthocyanins Figure 2. Simplified diagram of different strategies for process integration using aqueous
two-phase systems: (1) Extractive conversion, (2) Extractive fermentation, (3) Cell disruption
using ATPS PEG-dextran. The authors
integrated with product primary recovery, and (4) Extractive purification.
found that strawberry cells with a high
concentration of anthocyanins parti-
tioned differently from other strawber-
ry cells, causing the fractionation into two cell populations
with significantly different anthocyanin content. Regarding or-
ganelle fractionation, Morr and Morre [21] reported a meth-
od for the isolation of plasma membranes and Golgi apparatus
from cultured mammalian cells. Such a method exploits the
differences of charge and hydrophobicity of plasma mem-
branes in order to fractionate cell organelles. The authors
observed clear phase affinity differences among different mam-
malian cell membranes types, including endoplasmic reticu-
lum, mitochondria, Golgi apparatus, lysosomes and plasma
membranes.
3 Process Integration
The increasing need for biotechnological industries to develop
new products, has focused research to establish methods of
product recovery that integrate effectively with upstream cell
cultures to rapidly yield products in a suitable state for valida-
tion, formulation and delivery. The approach of process inte-
gration attempts to achieve specific objectives not efficiently
met by discrete unit operations by combining two or more
into one process. In this context, the application of ATPS for
process integration represents an attractive alternative for the
recovery of biological products. Four major areas of research
regarding process integration using ATPS can be identified: (1)
extractive conversion, (2) extractive fermentation, (3) cell dis-
ruption integrated with product primary recovery, and (4) ex-
tractive purification. A simplified representation of these four
areas is depicted in Fig. 2.
Extractive conversion is related to the recovery of biomole-
cules produced inside the ATP extraction system. In this ap-
proach a biochemical reaction takes places in one of the two
phases of the system while partition and recovery of the prod-
uct of interest occurs in the opposite phase. The continuous
removal of the product of interest favors the reaction equilibri-
um toward product formation. Extractive fermentation is a
strategy that allows the removal of the product of interest from
the fermentation broth as it is formed and excreted by the ex-
pression system. This approach helps to overcome low product
yields in a conventional fermentation process, recovering the
product of interest in one phase while the expression system
continues producing at the opposite one. Although this ap-
proach has proven to have great potential for process integra-
tion [22], restrictions regarding compatibility between the ex-
pression system and the ATPS composition must be taken into
consideration. Extractive fermentation can only be used when
the product of interest is secreted by the expression system,
i.e., when it is extracellular. When the product of interest is in-
tracellular, cell disruption is required in order to release the
product. In this context, the application of ATPS for the inte-
gration of cell disruption and primary recovery represents an
innovative approach. Studies have shown the potential of this
strategy, in which cell disruption of the expression system and
separation of the product of interest from contaminants (in-
cluding cell debris) are achieved in a single step [23]. Although
ATPS is considered a primary recovery technique, it is also ca-
pable to partially purify the product of interest. The use of
chemically modified polymers for phase formation and the ad-
dition of affinity ligands result in an increase of the selectivity
of the extractive system [2]. It is clear that, the potential of
ATPS, to purify a product of interest, increases considerably
when both extraction and purification are integrated in one
single step. Extractive purification using ATPS must be fol-

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Chem. Eng. Technol. 2008, 31, No. 6, 838845
lowed by polymer removal, which is usually achieved by means
of ultrafiltration.
4 Novel Approaches for Downstream
Processing using ATPS
4.1 Molecular Characterization of Proteins
Partitioning in aqueous two-phase systems (ATPS) is basically
a surface-dependent phenomenon. It is a process where the ex-
posed residues of the protein come in contact with the phase-
forming components [24]. Several protein properties influence
this behavior, e.g., charge, size, hydrophobicity, and solvent af-
finity. Under controlled conditions, and using appropriate
well-characterized ATPS, two-phase partitioning can be
exploited to discriminate between similar proteins based on a
single molecular property such as size or hydrophobicity.
Most of the reports on the use of ATP partitioning describe
it as a practical technique that allows recovery and purification
of biological products from a variety of sources. However, this
technique is also useful in understanding the chemical proper-
ties and behavior of proteins in solution. From these proper-
ties, hydrophobicity plays an important role during partition-
ing especially for systems with high molecular weight
polymers or high salt content [25]. The use of aqueous two-
phase systems (ATPS) to measure the functional hydropho-
bicity of proteins has been previously reported by several
authors using a wide range of polymers and salts [5, 2628].
This functional hydrophobicity is a measure of the real surface
hydrophobicity of the protein, and it is the result of the direct
interaction between the exposed residues on the surface of the
protein and the solvent molecules.
It has been stated previously that the establishment of ade-
quate downstream strategies requires the characterization of
the contaminant proteins from crude extracts [29]. It is clear
that a better understanding of the molecular characteristics of
the potential contaminants, e.g., MW distribution, hydropho-
bicity, and pI, etc., will be beneficial for the selection, optimi-
zation and design of the downstream strategies.
4.2 ATPS and 2D Electrophoresis for 3D
Characterization of Proteins
A recent application of PEG-salt ATPS (including PEG 3350 or
higher) includes coupling with proteomic tools, e.g. two-di-
mensional electrophoresis, 2-DE, in order to exploit the versa-
tility of two-phase partitioning to fractionate crude extracts to
eliminate large protein fractions as major contaminants based
on a single property like molecular size or hydrophobicity. The
coupling of 2-DE with other analytical techniques has been re-
ported to overcome some of the drawbacks of SDS-PAGE, e.g.,
difficulties in detecting low abundance proteins, aggregation of
proteins, particularly hydrophobic proteins and reproducibili-
ty issues, by adding a preliminary analytical step. Some exam-
ples include a non-denaturing anion exchange chromatogra-
phy prior to 2-DE to simplify the proteome and detect
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Aqueous Two-Phase Systems 843
functional associations between proteins [30], and a pre-frac-
tionation/concentration step using affinity partitioning in
ATPS prior to 2-DE-LC/MS for membrane proteins enrich-
ment [31]. It has been recently reported that optimized ATPS
can serve as a presorting stage in proteomic studies since it can
be customized for selective extraction and/or partition of large
fractions of proteins from crude extracts. These recent applica-
tions include 3D protein characterization when coupled with
2D electrophoresis [26, 32].
Two-dimensional gel technology offers protein visualization
for a variety of purposes, in addition to allowing empirical es-
timation of molecular weight, pI and expression levels [33].
The use of this information coupled with aqueous two-phase
partitioning was first reported by Gu and Glatz [26] with the
aim of obtaining a protein profile from corn endosperm. A
three-dimensional pattern emerged from plotting MW, pI and
a hydrophobicity scale obtained from ATP partitioned corn
proteins. Valuable information on the properties of potential
contaminant proteins was obtained with this experimental ap-
proach on route to facilitating further downstream purifica-
tion. Optimized ATPS can also be used to increase solubiliza-
tion of highly hydrophobic proteins, which has been one of
the main drawbacks of 2-DE-based proteomic protocols. In
this context, proteome data and ATPS can provide valuable
tools to help define properties of a recombinant protein that
can be exploited in order to facilitate extraction and/or purifi-
cation.
4.3 ATPS and Dielectrophoresis
Dielectrophoresis (DEP) is a non-destructive electrokinetic
transport mechanism, with important applications in biose-
parations. DEP is the movement of particles in a nonuniform
electric field, and it can occur in AC or DC electric fields. DEP
has been successfully applied for the manipulation of a wide
range of particles: biomolecules [3438], virus [3941], bacte-
ria [4249], spores [50, 51], mammalian cells [5254] and
parasites [5557]. DEP offers the possibility of sample concen-
tration and separation in a single step. Due to polarization
effects in nonuniform electric fields, particles of interest are
dielectrophoretically immobilized and can be significantly
concentrated almost to solid density. Therefore, DEP is a tech-
nique that can be employed in the polishing stages of a biopro-
cess [45].
ATPS have tremendous potential for the primary recovery
and characterization of many high value products ranging
from small phytochemicals to virus-like particles [1]. The
combination of ATPS with dielectrophoresis offers a novel
strategy where bioparticles can be separated employing ATPS,
and then selectively concentrated by employing DEP. The mar-
riage of these two powerful techniques has unlimited applica-
tions, from the extraction and concentration of important bio-
particles such as nutraceuticals and pharmaceuticals, to the
recovery and re-circulation of microorganisms employed in a
bioreactor system.
Additionally, both techniques are being employed for bio-
particle characterization, i.e., particles are being characterized
in terms of their thermodynamic [2729] and physicochemical
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844 J. Benavides et al.
properties employing ATPS, and in terms of their dielectric
properties by employing DEP [38]. Integration of ATPS and
DEP can provide a system for two-dimensional characteriza-
tion of biological particles that should provide guidelines for
the bioprocess design.
5 Future Trends and Challenges
It has been generally recognized that because the purity re-
quirements vary, the recovery and purification, i.e., biosepara-
tion of bioproducts, represents the limiting steps in process de-
velopment [58]. The biotechnology industry faces challenges
and trends in bioseparation today that are different from those
that existed a decade ago. The reason for this is mainly due to
the considerable increase in product concentration in the fer-
mentation broth. This is the result of the optimization of re-
combinant protein production. The final concentration of the
product and yield are virtually hundreds of times higher than
they were 15 years ago [59]. Furthermore, the urgent need to
develop bioseparation processes to obtain low molecular
weight non-protein products will demand new bioengineering
strategies. In this context, ATPS based strategies represent an
attractive alternative for consideration. The potential interac-
tions of ATPS with novel techniques, i.e., dielectrophoresis and
2-DE, Fig. 3, will favor the impact of the approach on product
characterization and process development. The increment of
studies related to the characterization and development of
ATPS recovery processes for these particular type of com-
pounds, mainly phytochemicals and other small molecules
with nutraceutical properties, will be required. It is expected
that the challenge that industries face relating to the handling
of concentrate feedstock will benefit the adoption of ATPS-
based strategies. The potential continuous operation of ATPS
for in situ product recovery will draw attention to the develop-
ment of ATPS recovery processes at a commercial scale. In par-
Figure 3. Potential interactions between novel techniques and
ATPS for bioproduct characterization and process development.
2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eng. Technol. 2008, 31, No. 6, 838845
ticular, the requirements for the recovery of a variety of biolog-
ical products with increasing significance for the food,
cosmetic, textile, detergent and health industries, will be ad-
dressed. An important future application of ATPS involves the
use of this technique as a sample preparation stage prior to
analysis for clinical and research purposes. Detection and
quantification of biomarkers via proteomic analysis represents
a good example of this area of application. It can be antici-
pated that, in the near future, novel bioengineering approaches
and techniques will become available to address the unat-
tended issues of the recovery and purification bioproducts. It
is also anticipated that ATPS will play a major role in the de-
velopment of some of these strategies.
6 Conclusions
The challenges and trends in bioseparations faced by biotech-
nology today and in the near future are different from those
that existed a decade ago. Although, the potential of ATPS for
the recovery of biological products has been proven, the tech-
nique has not been widely exploited at a commercial scale. The
reluctance of industries to adopt ATPS processes has been at-
tributed in some degree to the time involved in the learning
process for the technique and the poor understanding of the
mechanism governing partition of solutes in the systems. Prac-
tical experience derived from the development of ATPS pro-
cesses generates knowledge concerning the molecular events
governing partition behavior. In this review, the importance of
ATPS for the recovery and partial purification of biological
products was highlighted. The benefits of ATPS in process in-
tegration have also been discussed. In the route to facilitate the
establishment of bioseparation process conditions, the poten-
tial of the novel approach using ATPS coupled to two-dimen-
sional (2D) electrophoresis and dielectrophoresis for the physi-
cochemical characterization of biomolecules and process
development was raised. The review presented herein is con-
sidered to be a relevant contribution to facilitate the establish-
ment of ATPS processes at a commercial scale.
Acknowledgements
The authors would like to acknowledge the financial support
of Tecnologico de Monterrey Research Chairs (Grant Nos.
CAT080 and CAT005).
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