AQUEOUS TWO-PHASE SYSTEMS (ATPS)
overview of ATPS applied to the extraction/purification
DANIELA DE ARAÚJO VIANA
MARQUES1 , JOÃO VITOR
DUTRA MOLINO1 , PRISCILA
GAVA MAZZOLA2 , MARIA S.
VICCARI GATTI2 , and
ADALBERTO PESSOA JÚNIOR1
1
University of São Paulo, São
Paulo, SP, Brazil
2 University of Campinas,
Campinas, SP, Brazil
INTRODUCTION
Biopharmaceuticals approved for marketing have greatly
improved the treatment in the case of many disease groups
such as cancer, diabetes, growth disorders, hemophilia,
and hepatitis. The global pharmaceutical market is pre-
dicted to reach US$ 1.3 trillion in 2020 (1). The processes
for producing these biopharmaceuticals achieved great
advances, increasing productivity and income with the use
of improved culture media and innovative strategies (2,3).
However, purification processes have been neglected, caus-
ing a bottleneck in the production of biopharmaceuticals
when compared with bioproduct production, and also have
a higher cost, achieving values from 50–80% of the total
production costs (4). Therefore, it is essential to develop
new approaches to improve separation and purification
efficiency while maintaining the overall process econom-
ically viable and meeting the quality standards required
for the market (5).
Aqueous two-phase systems (ATPSs) have been used to
develop bioprocesses for the recovery and purification of
many biological products including proteins, genetic mate-
rial, bionanoparticles, cells, and organelles (6,7). Notable
examples include using ATPSs to remove color from tex-
tile wastewaters (8), metal ions (9), organic pollutants
from the environment (10), and aromatics from crude oil
(11). Another example is the preconcentration of mam-
malian genomic DNA before analytical techniques, which
would improve lower detection limits, thereby enhancing
biomarker detection. It could aid cancer detection in early
stages, for instance, thus increasing survival rates (12).
The ATPS is an interesting alternative to the current
downstream bottleneck because it has interesting features
such as comparatively low energy and is less time consum-
ing, besides having low material cost. In addition, it can
also be designed for continuous operation, owing to the low
toxicity of phase-forming chemicals and biocompatibility,
which ensure a high yield (7). It is also able to integrate
clarification, concentration, and purification in a single
operation unit and, as it is a liquid system, it can be easily
scaled up, increasing the volume of components (13). In
Encyclopedia of Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, edited by Michael C. Flickinger
© 2013 John Wiley & Sons, Inc. Published 2013 by John Wiley & Sons, Inc.
this work, an attempt has been made to present a general
of biomolecules and bioparticles and the biotechnology
field, focusing on therapeutic proteins, genetic materials,
virus-like particles, and viruses, in which ATPSs might
make an important contribution (Fig. 1).
ATPS TECHNOLOGY DESCRIPTION
Liquid/liquid systems composed mostly of water in
both phases are denominated ATPSs. In this review,
we focus on three types of ATPSs used in extractions
of biomolecules: systems formed by polymer/salt, poly-
mer/polymer, and systems formed by surfactants (cloud
point systems) (Fig. 2).
Mechanisms related to the separation of two aqueous
phases, partition behavior of solutes and particles between
such phases, are dominated by the system in thermody-
namic equilibrium. There are several types of ATPSs based
on the chemical nature of their main constituents (14–16).
Specific chemical interactions involved in phase formation
in ATPSs highly depend on the type of ATPS (17).
Moreover, the separation of biomolecules through this
system is complex, as the component partition is influenced
by short- (van der Waals) and long-range electrostatic
molecular interactions (13) between the biomolecule and
the surrounding phases (18). The other driving force in
ATPS partition is the excluded volume effect, which is
important for larger targets such as bioproducts (19).
ATPS Technology Compared with Other Techniques
Conventional liquid/liquid extraction, using organic/
aqueous phase systems, is a classical and versatile
technology that has been established as a workhorse in
the pharmaceutical industry. However, in spite of all
its advantages, this unit operation has not gained wide
industrial recognition in the field of biotechnology owing
to its poor solubility and possible protein denaturation in
organic solvents (20); therefore, its use in biotechnology
has been limited to the recovery of low molecular weight
products, such as antibiotics and organic acids from
fermentation broths (5). ATPSs are more suitable as
a downstream processing technology of bioproducts,
particularly for continuous operation (7,21,22).
Chromatography Techniques. Despite the essential role
of packed bed chromatography owing to its simple and high
resolving power, high titer processes have been imposing
practical limitations, suggesting that the true bottleneck
relies on the first adsorptive column. With an increasing
scale, very large columns are needed; thus, the perfor-
mance of the process decreases as additional costs of resins,
buffers, and other consumables outstrip any savings made
by increasing the productivity (5,23). Examples of sim-
ple nonchromatographic bio-separation technologies that
1
2 AQUEOUS TWO-PHASE SYSTEMS (ATPS)

procedure is time consuming (105-min first run and 18-h

Biomolecules/ Other ATPSS
applications second run). Long periods of time during the processing
bioparticles in biotechnology can affect the infectivity of the purified product. Previ-
Therapeutic proteins ous studies have shown that the viral infectivity decreases
Genetic material
Viral particles approximately 45% when compared with the original value
after a density gradient centrifugation (27). Density gra-
dient centrifugation is also difficult to scale up at both
ATPS extraction systems types: laboratory and industrial scales.
Among other techniques used to recover adenoviral
vectors are some chromatographic types, including
ATPS ATPS size-exclusion chromatography (28), affinity chromatogra-
ATPS
phy (29), and anion-exchange chromatography (30). These
Polymer/salt Cloud point
Polymer/polymer techniques are almost limited because they commonly fail
to discriminate the infective particles and contaminants
such as noninfective particles and particle components.
ATPS might possess a volumetric capacity (occupancy
per phase volume) for adenoviral vectors higher than
Figure 1. General overview of this review. the density gradient centrifugation and adsorptive
chromatography (31–33), which means that ATPSs
can handle higher particle numbers in a production
Polymer/ Polymer/ Cloud advantage.
salt polymer point
Microemulsion Extraction Method. Microemulsion is
composed of water, organic solvent, and surfactant,
occasionally with an alcohol as a cosurfactant. The
multiphase microemulsion systems were first described
by Winsor. Two-phase systems, called Winsor I and
Winsor II, correspond to oil in water microemulsion
(Wm) coexisting with an excess oil phase and water in
oil microemulsion (Om) coexisting with an excess water
phase, respectively. A Winsor III system corresponds
to concentrate surfactant into a bicontinuous phase (D)
coexisting with excess oil and excess water. Sometimes,
water, surfactant, and organic solvent form a single phase,
which is also called Winsor IV. The type of microemulsion
is determined by the compositions, temperature, and
volume ratio of oil to water (34).
A strategy for separation of a L-phenylacetylcarbinol
(PAC) product and recovery of a nonionic surfactant in the
microbial transformation broth with Winsor I microemul-
sion was developed by Wang et al. (34). The product
recovery efficiency was 76.9% and the nonionic surfac-
Figure 2. Schematic representation of the three ATPSs reviewed. tant with low HLB (hydrophile–lipophile balance) value
was limited, only 66.5%.
Gomez del Rio and Hayes (35) studied the extraction of
have been revisited recently include flocculation, precipi- different proteins (bovine serum albumin, α-chymotrypsin,
tation, liquid/liquid extraction, and crystallization (5,24). cytochrome c, and lysozyme) in aqueous solution by
Aguilar et al. (25) compared the ATPS with the isooctane solution that contained a surfactant mixture
ion-exchange-based chromatography process for penicillin (Aerosol-OT, or AOT, and a 1,3-dioxolane (or cyclic ketal)
acylase (PA) purification from Escherichia coli. In this alkyl ethoxylate, CK-2,13-E5.6), producing a three-phase
study, higher extraction yields were achieved (97% vs 48%, (Winsor III) bicontinuous microemulsion, a phase highly
respectively) and a predicted operation cost reduction of concentrated in protein (5–13 g/L), and small in volume
approximately 37%, even without PEG, and phosphate (12.2% of entire volume). Greater than 90% forward
recycling, which would reduce further costs and process extraction was achieved. The driving force for forward
steps from 7 to 4. However, a lower purification factor was extraction was mainly electrostatic attractions imposed
achieved (3.5 vs 5.7). by the anionic surfactant AOT, with the exception of BSA
Purification of adenoviral vectors is another example at high ionic strength, which interacted via hydrophobic
that normally consists of three steps to obtain the highest interactions. Through the use of aqueous stripping
purity, including clarification by conventional centrifu- solutions of high ionic strength (5 wt%) and/or pH 12.
gation, followed by two-step banding in cesium chloride (to negate the electrostatic attractive driving force),
gradients (26). However, the chemical cost is high and the cytochrome c and α-chymotrypsin were back-extracted

from the middle phase at >75% by mass, with the specific
activity of recovered α-chymotrypsin being >90% of its
original value.
Ionic Liquid-Based Aqueous Two-Phase System. In the
past few years, Gutowski et al. (36) demonstrated that the
addition of inorganic salts to aqueous solutions of ionic liq-
uids (ILs) can cause liquid/liquid mixing and induce ATPS
(ionic). Owing to their relatively high content of water
and nonuse of volatile compounds, the IL-based ATPSs
are friendly to the environment, and therefore have been
used to extract/separate amino acids, drugs, low molecu-
lar mass compounds, radiological isotopes, proteins, and
enzymes (37).
Louros et al. (38) studied the extraction of
L-tryptophan, β-carotene, rhodamine 6G, and caffeine
solutes using a phosphonium-based ATPS. High-partition
coefficients were obtained with hydrophobic biomolecules
such as β-carotene. The authors observed that the
partitioning behavior of all studied biomolecules follows
the hydrophilic/lipophilic balance between the ILs and
biomolecules. ATPS-ILs based on [BMIM][Cl] have
been also used for the extraction of testosterone and
epitestosterone from human urine by ATPSs with
extraction efficiencies of 80–90% for both analytes (39).
The extraction of proteins by IL-based ATPSs was
first realized by Du et al., who extracted proteins from
human body fluids by employing a [C4MIM][Cl]/K2 HPO4
system (40)
Proteins such as BSA, trypsin, papain, and lysozyme
were chosen as model proteins, and their partition behav-
ior in the ATPSs of cholinium IL (cholinium acetate, cholin-
ium propionate, cholinium butyrate, cholinium glycolate,
cholinium lactate, di-cholinium oxalate, or tri-cholinium
citrate) + PPG400 was examined. The cholinium IL +
PPG400 ATPSs can be used to extract proteins efficiently,
and the extraction efficiency decreases with the increase
in the size of proteins and the tie-line length of the ATPSs.
(37).
The extraction of BSA, trypsin, cytochrome c, and
γ -globulins in IL-based ATPSs was demonstrated to be
effective using ILs. It is found that 75–100% of the proteins
can be extracted into the IL-rich phases. The extraction
efficiency of cytochrome c is decreased slightly with the
increase in pH values. The extraction efficiency of the BSA
increases with the increase in both alkyl chain length of the
ILs and temperature of the systems. More than 88–90% of
the activity was maintained in the ionic-liquid-rich phase
of the ATPSs. Compared with the traditional PEG ATPS,
the IL-based ATPSs have the advantages of lower viscos-
ity, little emulsion formation, quick phase separation, and
so on (41).
The IL-ATPS has been applied also to extract antibi-
otics such as penicillin G (42), amoxicillin, and ampicillin
(43). Jiang et al. (44) had separated penicillin from the fer-
mentation broth by [C4mim]BF4-inorganic salt IL-ATPS
and imidazole-terminal PEG (I-PEG)-Na2HPO4 ATPS
(45).
AQUEOUS TWO-PHASE SYSTEMS (ATPS) 3
ATPS AS AN EXTRACTION AND PURIFICATION METHOD
FOR BIOLOGICAL MOLECULES AND PARTICLES
The ATPS for bioprocess development has focused mainly
on proteins, that is, enzymes, therapeutic proteins includ-
ing antibodies, colored proteins (46), α-interferon, as well
as blood-serum-related proteins (47,48). Conversely, plas-
mid DNA (49–52), virus-like particles (53), and purifica-
tion of adenoviral vectors (31) by ATPSs were also used.
The ATPS confers a mild extraction environment with
a considerable advantage over other recovery strategies,
for example, using solvents. The use of solvents can neg-
atively 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 purification) is
always a major concern (46).
Biomolecules Extraction and Purification by Polymer/Salt
Systems
The economic and environmental sustainability of an
ATPS-based capture process for a lot of biomolecule extrac-
tion and purification is considerably advantageous in
terms of process economy and operation.
Platis and Labrou (54) reported the primary recovery
and partial purification of the antihuman immunodefi-
ciency 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, a purification factor of 3–4, and a purity
of approximately 1.5% were achieved.
Lee and Forciniti (55) purified an immunoglobulin
gamma (lgG) fully unglycosylated from corn meal. Lower
yields were reached when compared with results reported
by Platis et al. (56), which could be explained by the dif-
ferences in the production source, such as bacteria and
mammalian cells, respectively. In this study, Lee and
Forciniti obtained a purification factor of 20, an antibody
purity of 72%, and a total process yield of 50%.
Azevedo et al. (57) achieved promising results using
hybridoma supernatant and a system composed of
PEG/citrate to IgG extraction. The authors obtained
a recovery yield of 99%, protein purity of 96%, SEC
(size-exclusion chromatography) purity of 76%, and
purification factor of 6. Back-extraction was performed
using NaCl to force the antibody to the PEG-rich phase
in the first step and to add a fresh citrate phase in the
second step, thus removing the antibody from the polymer
phase (58). This shows the adaptability of ATPSs for
extraction, by directing target molecules to the desired
phase through a manipulation of the system’s conditions.
The enzyme α-galactosidase has several potential appli-
cations, including treatment for Fabry disease. The par-
tially purified enzyme was obtained from Aspergillus
oryzae in a primary step using 12% (w/w) PEG 4000,
and 11.9% (w/w) KH2 PO4 –K2 HPO4 at system pH 5 and
temperature of 25 ◦ C. A comparison between a purifi-
cation process using ion exchange chromatography (IEC)
4 AQUEOUS TWO-PHASE SYSTEMS (ATPS)
with a previous acetone fractionation, and ATP extraction,
demonstrated a better overall recovery in ATPSs (11.5%
vs 87.6%, respectively). This low result achieved with IEC
might be related to the use of an organic solvent in the
initial steps, denaturing the target enzyme.
Interleukin-18-binding protein (IL-18BP) has been sug-
gested as a therapeutic protein in a number of dis-
eases and disorders, such as psoriasis, Crohn’s disease,
rheumatoid arthritis, psoriatic arthritis, liver injury, sep-
sis, atherosclerosis, and allergies. Kornmann and Baer (59)
performed the IL-18BP extraction in PEG/sulfate ATPS.
The best performing ATPS was composed of 11.25% PEG
10000/11.25% Na2 SO4 at pH 5. This ATPS allowed a recov-
ery of 98% of IL-18BP in the PEG-rich phase, with a final
purity of 92% (59).
Extraction and Purification of Bioparticles by Polymer/Salt
Systems
ATPS is described as a different approach for initial purifi-
cation of plasmid DNA (pDNA) from main contaminations,
consisting in a promising alternative for chromatographic
methods (15,49,60,61). Gomes et al. (50) used ATPSs to
partially purify pDNA from E. coli alkaline lysates, based
on PEG 600, and a mixture of 25% (w/w) of ammonium
sulfate and 75% (w/w) of sodium citrate; it offered a good
compromise between plasmid recovery (91.1%) and purity
(17.2%) (50).
The importance of virus-like particles (VLP) and viruses
in biotechnology has recently increased, as the first can
be used as scaffolds or as vaccines (62,63) and the second
as active viral subunit vaccines, vectors for gene therapy
(52), and to transfer genes in order to establish stable cell
lines (64).
The recovery of B19 virus-like particles using the ATPS
was studied by Luechau et al. (53). First, a process was
designed to recover B19 particles from the bottom phase
of a PEG 1000-magnesium sulfate system while removing
cell debris and 31% of total proteins in the upper, interface,
and sediment phases. When it came to the analysis of VP1
and VP2 capsid proteins after extraction, the yield of
B19 particles in the bottom phase was 92.8% or 85.7%,
respectively (53).
Benavides et al. (65) reported the use of PEG–
phosphate ATPSs to recover and partially purify
double-layered rotavirus-like particles (dlRLP) produced
by the baculovirus-insect cell expression system, using
ATPS PEG–potassium phosphate. A two-stage ATPS
consisting of PEG 400-phosphate with a 13.0 Vr (volume
of top phase/volume of bottom phase) and a 35% tie-line
length (TLL, w/w) at pH 7.0 provided with the best
conditions, intracellular dlRLP accumulated in the top
phase (recovery of 90%), whereas cell debris remained
in the interface. Furthermore, dlRLP from culture
supernatants accumulated preferentially in the interface
(recovery of 82%), using ATPSs with a Vr of 1.0, pH of
7.0, PEG 3350 (10.1%, w/w) and phosphate (10.9%, w/w).
The purity of the dlRLP from the culture supernatant
increased up to 55 times after using ATPS (65).
The potential of an alternative ATPS composed of PEG
300-phosphate was investigated for the recovery of ade-
noviral vectors. In this system, adenoviral vectors were
concentrated in the PEG-rich top phase, showing substan-
tially high recoveries, despite the complexity of the crude
viral lysate. It was evident that the chemical components
of ATPSs did not have a significant effect on the infec-
tivity of adenoviral vectors because a total recovery of
approximately 90% was obtained (31).
A miniaturization study on the recovery of bacterio-
phage T4 (initial titer 2.1 × 1011 pfu/mL) in ATPS PEG
8000-phosphate and PEG 600-sulfate at three different
scales (10 g, 2 g, and 300μL) was reported by Negrete et al.
(66). The results of partition behavior showed in the first
system with a Vr = 1.0 consist of a K greater than 5 with
infective particles (titer 108 –107 pfu/mL) concentrated in
the top phase. The bacteriophage T4 was concentrated in
the opposite phase in the PEG-600-sulfate system with
a consistent Vr = 0.8, K < 0.2, and infective particle
around 108 –105 pfu/mL for the scales analyzed. Unfortu-
nately, the chemical components of this particular system
(PEG/sulfate) had a negative impact on the bacteriophage
T4 infectivity (66).
Extraction and Purification of Biomolecules by
Polymer/Polymer System
Rosa et al. (67) reported the use of polymer/polymer ATPSs
in the presence of chemically modified (functionalized)
varieties of PEG for the recovery of human IgG pro-
duced by Chinese hamster ovary (CHO) cells. The authors
reported a 93% recovery yield and a 1.9 purification fac-
tor when PEG-dextran systems with modified 20% (w/w)
PEG 150-COOH were studied. A comparison with pure
PEG/dextran system demonstrated an improvement in
selectivity to IgG when this modified polymer was added.
In fact, an increase of 60-fold to selectivity was achieved.
PEG/dextran had a recovery yield of 28% and a purification
factor of 0.72 (67).
IgG recovery from the CHO supernatant was also eval-
uated using a system composed of PEG/dextran with
different functionalized PEGs (best ligands: glutaric acid
(GA), benzyl, and NH2 ). The best result was acquired
using PEG-GA as functionalized PEG. PEG/dextran sys-
tem resulted in a recovery yield of 23% and 39% protein
purity, and when PEG-GA was added, the recovery yield
increased to 97% and protein purity was of approximately
90% (68).
Extraction and Purification of Bioparticles by
Polymer/Polymer System
The distribution of bacteriophage T2, adenovirus, and
ECHO (enteric cytopathic human orphan) virus prototype
7 and 19 in ATPSs of sodium dextran sulfate, methyl-
cellulose, polyvinyl alcohol, and polyethylene glycol was
studied. By using an ATPS in one step, the virus parti-
cles may be concentrated 10–100 times. By using two-step
procedures, a concentration effect of about 1000 times has
been demonstrated (69).
Hazlett (70) studied the concentration of porcine
enterovirus strain T80 by separation in an aqueous
polymer two-phase system (PEG/sodium dextran sulfate),
which gave virus concentration factors of 56- to 105-fold
and recovery rates of 37–107% (70).

In 1982, Schloer and Breese investigated the purifi-
cation of malignant catarrhal fever virus (MCFV), using
two different ATPSs: PEG and dextran 10 or PEG and
dextran 70 at different concentrations in both cases. All
the partition coefficient values were <1, that is, MCFV
has been partitioned to the dextran-rich bottom phase
(recovery around 100%) (71).
In 1994, Gilljam and Hammar observed that the con-
ventionally applied centrifugation protocols for the con-
centration and purification of human immunodeficiency
virus type 1 (HIV-1) resulted in a low recovery of exter-
nal glycoprotein, gp120. Therefore, with a PEG/dextran
system gp120 and gag protein p24 used as a marker of
the virion, approximately 60% and 70% were recovered,
respectively, in 1% of the initial volume. The two proteins
were both about 30-fold purified and reverse transcrip-
tase activity and infectious titer were retained to a high
degree. The calculated molar ratio of gp120 to p24 was
twofold higher in the phase-extracted fraction than in the
material pelleted by ultracentrifugation (72).
Extraction and Purification of Biomolecules and Bioparticles
by Cloud Point Systems
Jozala et al. (73) demonstrated the application of the
cloud point system to recover nisin from fermented super-
natant using this type of system, which was mild to the
extraction of this biomolecule as observed by the activ-
ity balance obtained after the extractions (>96%) (73).
Another advantage of this type of system is its capability
to remove contaminants. Lopes et al. (74) demonstrate the
LPS removal efficiency of this system from a fermentation
broth of recombinant E. coli producing GFP. This system
was capable of, while extracting GFP at rates superior to
90%, removing more than 99,5% of LPS from the sample
(75).
Liu et al. (76) studied the partition behavior of bac-
teriophages (such as X174, P22, and T4) on micellar
ATPSs using n-decyl tetra (ethylene oxide) (C10 E4 ) as sur-
factant. The bacteriophages partitioned into the bottom,
on the micelle-poor phase of micellar system. The excluded
volume theory, which is able to successfully describe the
partitioning behavior of the smaller bacteriophage X174,
significantly overpredicts the partitioning behavior of the
larger bacteriophages P22 and T4 (76).
In a second work (part 1), the authors explained the
behavior of all three bacteriophages partitioned similarly
in function of temperature, and that the three had very dif-
ferent sizes (77). The authors concluded in the same work
(part 2), using bacteriophage P22 as an example, that the
measured viral partition coefficient at each temperature
decreased by about an order of magnitude when the vol-
ume ratio was decreased from 10 to 0.1, which clearly
indicated that entrainment is an important influencing
viral partitioning factor.
NEW TECHNIQUES INTEGRATED WITH ATPSs
Recent advances in experimental designs have led to the
increasing application of the ATPS to the microfluidic sepa-
ration of biomolecules. An important advancement related
AQUEOUS TWO-PHASE SYSTEMS (ATPS) 5
to the integrative microfluidic extraction has been also
investigated by combining an electric field and two-phase
laminar flow. These types of extractions, coupled with
external force, electric or magnetic, and affinity agents,
will certainly play an even more important role in the
next generation methods of microfluidic extraction-based
two-phase laminar flow systems (78,79).
Another process recently reported the integration of
ATPSs with 2D electrophoresis (80). Optimized ATPSs can
also be used to increase solubilization of highly hydropho-
bic proteins, which has been one of the main drawbacks of
2D electrophoresis-based proteomic protocols (46,81).
The other integrated process studied with ATPSs is
the extractive fermentation which allows a concurrent
extraction of toxic substrates and products, inhibiting
microorganism fermentation (82). Viana Marques et al.
(83) used PEG/phosphate salts ATPSs, integrating the
Streptomyces DAUFPE 3060 fermentation. The highest
clavulanic acid concentration was obtained in a bioreactor
PEG-rich phase with yield 30% higher than in the flasks,
thus demonstrating the potential of such a new process
for the production and extraction in the same step (83).
Pan et al. (84) applied a cloud point system in an extrac-
tive fermentation, using a combination of Triton X-114
and Triton X-45. They reported a 95% recovery yield of
lipase produced by Serratia marcescens. In this study,
researchers reached a concentration factor of 4.2-fold.
More interestingly, a fermentation process with 6% of
nonionic surfactant mixture (ratio of Triton X-114 to Tri-
ton X-45 was 4 : 1) presented the same microbial growth,
but a third increase in lipase production. This result could
be due to a more permeable membrane generated by the
presence of surfactants. Furthermore, cloud point systems
have high capability in removing endotoxin (LPS), the
major contaminant in bacteria fermentation. Lopes et al.
demonstrated 99% efficiency in removing LPS from GFP
produced in E. coli with 96% recovery yield (74). Fur-
ther coupling extractive fermentation studies with LPS
removal and product extraction would throw more light on
this subject, demonstrating its applicability.
CONCLUSIONS AND FUTURE TRENDS
In biopharmaceutical manufacturing, process develop-
ment represents 30% of the costs, an upstream processing
for 20%. However, the highest outlay in biopharmaceu-
tical manufacturing is attributed to the downstream
processing, which is responsible for a massive 40–70% of
the total costs incurred (85).
In this context, different types of ATPS strategies repre-
sent an attractive alternative to be considered. Two devel-
opment directions of ATPSs gradually emerge with ATPS
practical applications. First, costs of components should
be reduced and new cheap components developed to com-
prise ATPSs. Second, components should be reused and
recycled. The increment of studies related to the charac-
terization and development of ATPS recovery processes for
these particular types of compounds, therapeutic proteins,
genetic material, virus-like particles, and especially virus
6 AQUEOUS TWO-PHASE SYSTEMS (ATPS)
vectors will be required. Continuing discoveries in molecu-
lar biology and genetics provide the foundation for new and
improved processes and products in today’s biochemical
process industry. Efficient, cost-effective, scalable produc-
tion, and purification processes are thus needed to satisfy
the demands for therapeutic proteins, genetic material,
and viral vectors for therapeutic use and ATPSs may
contribute with this goal.
ACKNOWLEDGMENTS
This work was supported by FAPESP (São Paulo Research
Foundation, Brazil), CAPES (National Council for the
Improvement of Higher Education, Brazil), and CNPq
(National Council for Scientific and Technological Devel-
opment, Brazil).
REFERENCES
1. Biopharm International. The Case for Outsourcing Bio-
logics Process Development. Available at http://www
.biopharminternational.com/biopharm/article/articleDetail
.jsp?id=568930&pageID=1&sk=&date=. Accessed 2012
March 15.
2. Browne SM, Al-Rubeai M. Trends Biotechnol 2007; 25(9):
425–432.
3. Durocher Y, Butler M. Curr Opin Biotechnol 2009; 20:
700–707.
4. Roque A, Lowe C, Taipa M. Biotechnol Prog 2004; 20(3):
639–654.
5. Rosa PAJ, Ferreira IE, Azevedo AM, Aires-Barros MR. J
Chromatogr A 2010; 1217: 2296–2305.
6. Benavides J, Rito-Palomares M. J Chem Technol Biotechnol
2008; 83: 133–142.
7. Asenjo JA, Andrews BA. J Chromatogr A 2011; 1218(49):
8826–8835.
8. Huddleston JG, Willauer HD, Boaz KR, Rogers RD. J Chro-
matogr B 1998; 711: 237–244.
9. Rodrigue GD, da Silva MDCH, da Silva LHM, Paggioli FJ,
Minim LA, Reis Coimbra JSD. Sep Purif Technol 2008; 62(3):
687–693.
10. Xie S, Paau MC, Li CF, Xiao D, Choi MMF. J Chromatogr A
2010; 1217(16): 2306–2317.
11. Rogers RD, Willauer HD, Griffin ST, Huddleston JG. J Chro-
matogr B 1998; 711: 255–263.
12. Mashayekh F, Meyer AS, Shiigi SA, Nguyen V, Kamei DT.
Biotechnol Bioeng 2009; 102(6): 1613–1623.
13. Albertsson PA. Partition of cell particles and macro-
molecules: separation and purification of biomolecules,
cell organelles, membranes, and cells in aqueous polymer
two-phase systems and their use in biochemical analysis and
biotechnology. New York: John Wiley & Sons, Inc.; 1986.
14. Johansson HO, Feitosa E, Junior AP. Polymers 2011; 3:
587–601.
15. Kepka C, Rhodin J, Lemmen R, Tjerneld F, Gustavsson P. J
Chromatogr A 2004; 1024: 95–104.
16. Wang Z, Dai Z. Enzyme Microb Technol 2010; 46(6):
407–418.
17. Cabezas HJ. J Chromatogr B Biomed Appl 1996; 680(1–2):
3–30.
18. Zhi W, Song J, Bi J, Ouyang F. Bioprocess Biosyst Eng 2004;
27: 3–7.
19. van Roosmalen D, Lazzara MJ, van den Broeke LJP, Keuren-
tjes JTF, Blankschtein D. Biotechnol Bioeng 2004; 87(6):
695–703.
20. Krishna SH, Srinivas ND, Raghavarao KSMS, Karanth NG.
Adv Biochem Eng Biotechnol 2002; 75: 119–183.
21. Rosa PAJ, Azevedo AM, Sommerfeld S, Bäcker W,
Aires-Barros MR. J Chromatogr B 2012; 880(1): 148–156.
22. Vázquez-Villegas P, Aguilar O, Rito-Palomares M. Sep Purif
Technol 2011; 78(1): 69–75.
23. Gottschalk U. Biotechnol Prog 2008; 24: 496–503.
24. Rosa PAJ, Azevedo AM, Sommerfeld S, Bäcker W,
Aires-Barros MR. Biotechnol Adv 2011; 29(6): 559–567.
25. Aguilar O, Albiter V, Serrano-Carreón L, Rito-Palomares M.
J Chromatogr B 2006; 835: 77–83.
26. Laver W, Younghusband H, Wrigley N. Virology 1971; 45(3):
598–614.
27. McGrath M, Witte O, Pincus T, Weissman IL. J Virol 1978;
25(3): 923–927.
28. Huyghe BG, Liu X, Sutjipto S, Sugarman BJ, Horn MT,
Shepard HM, et al. Hum Gene Ther 1995; 6: 1403–1416.
29. O’Neil PF, Balkovic ES. Nat Biotechnol 1993; 11: 173–178.
30. Klyushnichenko V, Bernier A, Kamen A, Harmsen E. J
Chromatogr B 2001; 755(1–2): 27–36.
31. Negrete A, Ling T, Lyddiatt A. Process Biochem 2007; 42(7):
1107–1113.
32. Przybycien TM, Pujar NS, Steele LM. Curr Opin Biotechnol
2004; 15(5): 469–478.
33. Schügerl K, Hubbuch J. Curr Opin Microbiol 2005; 8:
294–300.
34. Wang Z, Xu JH, Liang R, Qi H. Biochem Eng J 2008; 41:
2–29.
35. Gomez del Rio JA, Hayes DG. Biotechnol Prog 2011; 27:
1091–1100.
36. Gutowski KE, Broker GA, Willauer HD, Huddleston JG,
Swatloski RP, Holbrey JD, Rogers RD. J Am Chem Soc 2003;
125: 6632–6633.
37. Li Z, Liu X, Pei Y, Wang J, He M. Green Chem 2012; 14:
2941–2950.
38. Louros CLS, Cláudio AFM, Neves CMSS, Freire MG, Mar-
rucho IM, Pauly J, Coutinho JAP. Int J Mol Sci 2010; 11:
1777–1791.
39. He CY, Li SH, Liu HW, Li K, Liu F. J Chromatogr A 2005;
1082: 143–149.
40. Du Y, Yu Y, Wang J. Chem Eur J 2007; 13: 2130–2137.
41. Pei Y, Wang J, Wua K, Xuan X, Lu X. Sep Purif Technol
2009; 64: 288–295.
42. Liu QF, Yu J, Li WL, Hu XS, Xia HS, Liu HZ, Yang P. Sep
Sci Technol 2006; 41: 2849–2858.
43. Soto A, Arce A, Khoshkbarchi MK. Sep Purif Technol 2005;
44: 242–246.
44. Jiang Y, Xia HS, Guo C, Mahmood I, Liu HZ. Ind Eng Chem
Res 2007; 46: 6303–6312.
45. Jiang YY, Xia HS, Yu J, Guo C, Liu HZ. Chem Eng J 2009;
147: 22–26.
46. Benavides J, Aguilar O, Lapizco-Encinas BH, Rito-Palomares
M. Chem Eng Technol 2008; 31(6): 838–845.
47. Lin YK, Ooi CW, Ramanan RN, Ariff A, Ling TC. Sep Sci
Technol 2012; 47(7): 1023–1030.

48. Selvakumar P, Ling TC, Walker S, Lyddiatt A. Sep Purif
Technol 2012; 90: 182–188.
49. Frerix A, Müller M, Kula MR, Hubbuch J. Biotechnol Appl
Biochem 2005; 42: 57–66.
50. Gomes GA, Azevedo AM, Aires-Barros MR, Prazeres DMF.
Sep Purif Technol 2009; 65: 22–30.
51. Rahimpour F, Feyzi F, Maghsoudi S, Hatti-Kaul R. Biotech-
nol Bioeng 2006; 95: 627–636.
52. Trindade IP, Diogo MM, Prazeres DM, Marcos C. J Chro-
matogr A 2005; 1082: 176–184.
53. Luechau F, Ling TC, Lyddiatt A. Food Bioprod Process 2011;
89(4): 322–327.
54. Platis D, Labrou NE. J Chromatogr A 2006; 1128: 114–124.
55. Lee JW, Forciniti D. Biotechnol Prog 2010; 26(1): 159–167.
56. Platis D, Drossard J, Fischer R, Ma JKC, Labrou NE. J
Chromatogr A 2008; 1211(1–2): 80–89.
57. Azevedo AM, Rosa P, Ferreira I, Aires-Barros M. J Biotechnol
2008; 132: 209–217.
58. Azevedo AM, Gomes G, Rosa PAJ, Ferreira F, Pisco AMMO,
Aires-Barros MR. Sep Purif Technol 2009; 65: 14–21.
59. Kornmann H, Baer G, (to Ares Trading S.A.). US Patent
7439336B2. 2008.
60. Luechau F, Ling TC, Lyddiatt A. Sep Purif Technol 2009;
67(1): 64–70.
61. Kula MR, Frerix A, Geilenkirchen P, Mu M. Biotechnol
Bioeng 2007; 96(1): 57–66.
62. Cardinale D, Carette N, Michon T. Trends Biotechnol 2012;
30(7): 369–376.
63. Parra GI, Bok K, Taylor R, Haynes J, Sosnovtsev SV,
Richardson C, et al. Vaccine 2012; 30: 3580–3586.
64. Gerard RD, Meidell RS. Trends Cardiovasc Med 1993; 3(5):
171–177.
65. Benavides J, Mena JA, Cisneros-Ruiz M, Ramı́rez OT, Palo-
mares LA, Rito-Palomares M. J Chromatogr B 2006; 842(1):
48–57.
66. Negrete A, Ling TC, Lyddiatt A. J Chromatogr B 2007;
854(1–2): 13–19.
67. Rosa PAJ, Azevedo AM, Ferreira IF, Vries JD, Korporaal R,
Verhoef HJ, et al. J Chromatogr A 2007; 1162: 103–113.
AQUEOUS TWO-PHASE SYSTEMS (ATPS) 7
68. Azevedo AM, Rosa PAJ, Ferreira IF, Pisco AMMO, Vries JD,
Korporaal R, et al. Sep Purif Technol 2009; 65: 31–39.
69. Philipson L, Albertsson PÅ, Frick G. Virologie 1960; 11(3):
553–571.
70. Hazlett DTG. Can J Microbiol 1977; 23(8): 1052–1058.
71. Schloer GM, Breese SS. J Gen Virol 1982; 58: 101–110.
72. Gilljam G, Siridewa K, Hammar L. J Chromatogr A 1994;
675: 89–100.
73. Jozala A, Lopes A, Mazzola P, Magalhães P, Penna TV,
Pessoa A Jr. Enzyme Microb Technol 2008; 42(2): 107–112.
74. Lopes AM, Magalhães PO, Mazzola PG, Rangel-Yagui CO,
de Carvalho JCM, Penna TCV, et al. Sep Purif Technol 2011;
81(3): 339–346.
75. Lopes AM, Magalhães PO, Mazzola PG, Rangel-Yagui CO,
Carvalho JCM, Penna TCV, et al. Biotechnol Prog 2010;
26(6): 1644–1653.
76. Liu CL, Kamei DT, King JA, Wang DI, Blankschtein D. J
Chromatogr B 1998; 711(1–2): 127–138.
77. Kamei DT, Liu C, Haase-Pettingell C, King JA, Wang DIC,
Blankschtein D. Biotechnol Bioeng 2002; 78(2): 190–202.
78. Huh YS, Jeon SJ, Lee EZ, Park HS, Hong WH. Korean J
Chem Eng 2011; 28(3): 633–642.
79. Yamada M, Kasim V, Nakashima M, Edahiro J, Seki M.
Biotechnol Bioeng 2004; 88(4): 489–494.
80. Roy I, Mondal K, Gupta MN. J Chromatogr B 2007; 849(1–2):
32–42.
81. Shevchenko G, Sjo MOD, Malmstro D, Wetterhal M,
Bergquist J. J Proteome Res 2010; 9: 3903–3911.
82. Lye GJ, Woodley JM. Trends Biotechnol 1999; 17: 395–402.
83. Marques DAV, Pessoa-Júnior A, Lima-Filho JL, Converti A,
Perego P, Porto ALF. Biotechnol Prog 2011; 27(1): 95–103.
84. Pan T, Wang Z, Xu JH, Wu Z, Qi H. Appl Microbiol Biotechnol
2010; 85(6): 1789–1796.
85. Research and Markets. Strategic Analysis of Downstream
Processing in Biopharmaceutical Production. Available
at http://www.frost.com/prod/servlet/report-brochure.pag?id
=F123-01-00-00-00. Accessed 2012 March 15.