Journal of Chromatography A, 1244 (2012) 1–13

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Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Review

Aqueous two-phase affinity partitioning systems: Current applications

and trends夽
Federico Ruiz-Ruiz, Jorge Benavides, Oscar Aguilar, Marco Rito-Palomares ∗
Centro de Biotecnología FEMSA, Departamento de Biotecnología e Ingeniería de Alimentos, Tecnológico de Monterrey, Campus Monterrey, Ave. Eugenio Garza Sada 2501 Sur,
Monterrey, NL 64849, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Aqueous two-phase systems (ATPS) have been studied and used for product recovery and purification
Received 15 December 2011 from diverse biological sources. ATPS are characterized by their versatility, easy scale up parameters,
Received in revised form 16 April 2012 process integration capability and relative low cost. This technique is commonly regarded as a primary
Accepted 25 April 2012
recovery stage mainly due to its low selectivity. However, the use of strategies involving the modifica-
Available online 7 May 2012
tion of ATPS with affinity ligands have resulted in significant increases in recovery yields and purification
folds of biological products. The aim of this review is to highlight current applications, trends and chal-
Keywords:
lenges regarding affinity partitioning in aqueous two-phase systems for the fractionation, recovery and
Aqueous two-phase systems
Affinity partitioning
purification of biological products.
Affinity ligand © 2012 Elsevier B.V. All rights reserved.
Aqueous two-phase affinity partitioning

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. Common chemical modification methodologies for polymer activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
3. Polymer–polymer affinity aqueous two-phase systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
4. Polymer–salt affinity aqueous two-phase systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
5. Affinity enhanced ATPAP systems using alternative phase components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
6. Trends and challenges of ATPAP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
7. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

the total bioprocesses costs [5,6]. Additionally, most of the unit

1. Introduction
operations comprising a biotechnology process are focused on
the recovery and purification of the product of interest. In this
There is a considerable interest in the development of efficient
context, the development of strategies for the optimization and
downstream processes for the recovery and purification of biolog-
design of efficient and economic downstream processes represents
ical products. The need to establish selective and scalable methods
a research area of great interest.
for product recovery has resulted in the implementation of diverse
Integration and intensification of unit operations are two
bioengineering strategies in the field. Nowadays, improvements
common strategies used for the optimization of downstream pro-
in liquid–liquid extraction systems, filtration devices and chro-
cessing. Process integration attempts to combine two or more unit
matography strategies allow the efficient recovery of natural dyes,
operations in one, reducing the total number of stages and increas-
antibodies, phytochemicals, proteins, virus like particles and other
ing recovery yields [2,7]. On the other hand, process intensification
products [1–4]. Purification strategies account for up to 80% of
is focused on maximizing the throughput in a given unit operation
by optimization of process parameters [2,7–9].
Aqueous two phase extraction is a liquid–liquid fractionation
夽 Presented at the 16th International Conference on BioPartitioning and Purifica-
technique that has been extensively used for the recovery and
tion, Puerto Vallarta, Jalisco, Mexico, 18–22 September 2011.
∗ Corresponding author. Tel.: +52 81 832800; fax: +52 818328 4136. purification of biological products. This technique involves the con-
E-mail address: mrito@itesm.mx (M. Rito-Palomares). struction of extraction systems that can be formed by two polymers,

0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.chroma.2012.04.077
2 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
a polymer and a salt, an ionic liquid and a salt, or a low molecular
weight alcohol and a salt mixed over a limit concentration thus
forming two immiscible phases [10,11]. Additionally, micellar and
reverse micelle aqueous two-phase systems (ATPS) can be formed
using solutions with ionic and/or non-ionic surfactants. Partition-
ing of biomolecules in ATPS depends on the choice of constituents
of the system, the concentration of the system constituents, the
volume ratio (VR ), the pH and temperature of the system [4,12].
Furthermore, the physicochemical properties (molecular weight,
superficial electrochemical charge, hydrophobicity, etc.) of the
solutes being partitioned on the systems also play a major role
on their partition behavior [4]. In general, ATPS have proven to be
suitable for the recovery of biological products from different ori-
gins [4,12,13]. However, one major drawback concerning the use
of ATPS is their lack of specificity. Although the manipulation of the
system parameters may favor the selective partitioning of the com-
pound of interest towards a particular phase, specificity usually is
not high enough to achieve complete separation between product
and contaminants.
Aqueous two phase affinity partitioning (ATPAP) consists of the
chemical modification of at least one phase forming component of
the system by attaching an affinity ligand with specificity for the
molecule of interest [14]. Therefore, selective interaction between
specific ligands in one of the polymeric phases of the system and the
protein, cellular fragment or biomolecule of interest followed by
their partition towards the modified polymer rich phase is achieved
[14]. In addition, affinity partitioning in liquid–liquid extraction
systems can also be achieved by the addition of free ligands (bio-
logical or chemical molecules) in solution to a traditional ATPS in
order to induce a partition shift of the desired target molecule
without the chemical modification of the phase forming compo-
nent [3]. ATPAP was discussed by Flanagan and Barondes [15] in
1975 when the selective partition of S-23 myleoma proteins in a
modified dextran–polyethylene oxide (PEO) system was studied.
The findings stated that the inclusion of polymer derivatives or
modified components to ATPS poses an advantage in downstream
processing of high value products. In addition, the authors con-
cluded that even though a marked increase in partition of the target
products using liquid–liquid affinity partitioning was observed, the
use of well-established chromatographic techniques may be con-
sidered, if needed, for the subsequent purification of the product
of interest. ATPAP provides a high compatibility system between
bioaffinity ligands and specific molecules, which can be manipu-
lated in order to develop more predictable purification strategies.
Therefore, it is possible to anticipate the partition coefficients for
a defined experimental system [16]. At preparative scale, ATPAP
systems also present important advantages for the development of
novel downstream strategies such as: high ligand concentration per
volume of polymer solution, which translates to high concentration
of bound target biomolecule in one of the system phases (process
intensification) and, fast equilibrium achieving [17]. In addition,
since ATPAP combines the bioselectivity of an affinity technique
with the robust conditions of traditional liquid–liquid extraction
systems, it is well suited for the specific recovery and purification
of diverse biological molecules.
In order to determine the potential of traditional ATPS for
the recovery and purification of biological molecules, experiments
characterizing the effect of system parameters such as polymer
molecular weight, tie line length (TLL), pH and VR are usually
evaluated [2,4]. In ATPAP, additional system parameters must
be considered in order to understand the partitioning behavior
of biomolecules. Based on the literature we have identified four
additional parameters usually studied on ATPAP: (1) optimum
pH level for product partitioning, (2) ionic strength of the sys-
tem in polymer–salt or polymer–polymer systems, (3) affinity
ligand nature and concentration and, (4) concentration of modified
polymers. These factors must be taken into consideration in order
to optimize ATPAP partition protocols since affinity interactions of
products and affinity ligands are highly dependent of the ionic state
of the active groups (according to pH level) and the ionic strength of
the system (depending on salt concentration) [3,5,15]. In addition,
ligand concentration and modified polymer concentration could
also prove to increase recovery and purification yields in ATPAP sys-
tems. Nevertheless, appropriate levels should be studied since high
ligand concentration could introduce shielding effects of ligands
toward target molecules and high modified polymer concentration
can generate steric hindrance problems between target molecules
and active receptor sites. These possible scenarios emphasize the
importance of the additional priorities that must be studied in
ATPAP systems, in order to design efficient and novel purification
strategies.
In addition to the traditional and empirical character of ATPS
design, new technical issues have to be considered within ATPAP.
At a first glance, partitioning of modified polymers with cou-
pled ligands or free ligands has to be addressed. First reports
employing ATPAP described that phase forming curves were not
greatly affected when modified polymers with coupled ligands
were present at low concentrations [15]. However, a recent inves-
tigation indicates that by increasing concentrations of a modified
polymer with a coupled ligand shifts the hydrophobic nature of the
polymer, due to the nature of the ligands. This shift may affect not
only the partition behavior of the solutes within the system but also
the phase formation behavior [18]. Another characteristic that may
be affected for the use of modified polymers is the ionic nature of
the polymer–ligand complex [18]. Since affinity coupling involves
very specific molecular interactions between a target molecule and
a specific ligand, the development of an ATPAP strategy must con-
sider the biological and chemical nature of the proposed affinity
ligand and target molecule. For example, affinity partitioning of
biological membranes has been achieved with ATPAP strategies in
which wheat germ agglutinin (WGA) has been coupled to phase
forming polymers. Due to the interaction between WGA with spe-
cific N-acetylglucosamine and sialic acid residues in the membrane
surface, partitioning was favored to a specific system phase [17]. In
this tenor, the chemical nature of both target products and ligands
must be understood as well as the degree of saturation (DS) of the
polymer (defined as the ratio of polymer/ligand molecules) in order
to maximize system efficiency [17]. In addition, polymer molecular
weight (MW) has an impact in product partitioning since volume
displacements are seen with high MW polymers [2,4,15]. In the
case of myeloma protein purification [15], the authors stated that
a 3-fold increase in polymer MW could enhance the Kp in 3.5-fold.
Also important is the hydrophobic character of the target molecule
and affinity ligand, since increases in polymer MW tend to enable
hydrophobic environments [4], and if this characteristic is not well
addressed the ATPAP strategy could be inefficient. These character-
istics must be considered when developing ATPAP models. Previous
reports have described ATPAP characteristics and particularly have
focused on the process integration capabilities of these systems.
Early researches addressed the recovery and purification of macro-
molecules like proteins. Then, the selective potential of ATPAP
systems was employed for separation of complex particles such
as cellular fragments and cells. In recent years there has been an
important growth of ATPAP based strategies, with new affinity lig-
ands that allow selective purification of high value molecules like
monoclonal antibodies or nucleic acids. In general, the full poten-
tial of this technique for the recovery and purification of biological
products requires further attention.
This report focuses in presenting a practical overview to high-
light the importance of ATPAP for the recovery and purification
of biological products. Common chemical modification method-
ologies for polymer activation are presented. A critical analysis
 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
of selected ATPAP systems reported in literature is summarized
to establish the benefits of the technique. Current challenges and
potential trends of the practical application of ATPAP for the recov-
ery of biological products are discussed.
2. Common chemical modification methodologies for
polymer activation
Affinity partitioning in ATPS provides a high-resolution tech-
nique that can be coupled to traditional polymer–polymer and
polymer–salt systems. In addition, aqueous two-phase micellar
systems have been modified in order to design new liquid–liquid
extraction processes with enhanced product affinity. Fig. 1 presents
the two most common strategies for the creation of ATPAP. It
should be noted that polyethylene glycol (PEG) and/or dextran
(DEX) have to be chemically active. Active polymers are charac-
terized by a chemical reaction of an activator molecule with a
polymer that renders increased activity to the functional groups
present in PEG or DEX. This methodology allows further covalent
coupling of affinity ligands. Nevertheless, as presented in Fig. 1,
recent researches emphasize the use of ligands free in solution
in order to avoid chemical activation of polymers. However, the
implementation of this strategy involves additional considerations
such as partitioning behavior of the free ligand in ATPS prior to the
analysis of the potential interaction of the ligand with the target
molecule.
A high diversity of molecules can be bound to ATPS poly-
mer components as affinity ligands [15,17]. Several activation
techniques have been developed in order to achieve high ligand
coupling. In the particular case of PEG and DEX, the strategies focus
on the active hydroxyl groups present in the polymers. Examples
of traditional procedures for polymer activation for ATPAP are pre-
sented in Table 1.
ATPAP purification strategies should consider the overall pro-
cessing time in the selected activation technique, the chemical
nature of the active groups in the polymers and the post-
activation ligand coupling to the polymer matrix. Considering the
proposed protocols in Table 1, ethylenediamine and butanediol
diglycidyl methodologies are characterized by multistep reactions
that require 65–110 h in order to obtain an active polymer. The
active hydroxyl groups in PEG present a chemical nature that allows
the further addition of nucleophilic groups present in proteins,
peptides and aminoacids [15,17,19,20]. In addition, the principal
advantage of these techniques involves the length of the spacer
arm or linker between the polymeric matrix and the affinity ligand
to be coupled. Since the linker arms with these protocols are longer
(7–15 Å), the interaction probability between an affinity ligand and
a particular molecule increases, generating a more efficient sepa-
ration process. Nevertheless, it should be noted that the extended
protocol time and relative complexity within these activation tech-
niques may limit, in some extent, their use in ATPAP.
Periodate and tresyl-chloride protocols exhibit shorter times
in terms of polymer activation. This characteristic is the princi-
pal advantage of the proposed methodologies, since 24 h or less
are required to generate an active polymer for the subsequent
ligand coupling stage. The active groups allow nucleophilic sub-
stitutions and additions, resulting in protein, carbohydrate binding
modules (CBM’s) and cofactors easy coupling [17,21–23]. Despite
the proposed advantages, spacer arms between the polymer and
the affinity ligand are shorter when compared with the ethylene-
diamine and butanediol diglycidyl methodologies. This diminishes
the complex flexibility, which in turn lowers the possible inter-
actions with the target molecules in the system. Nevertheless, it
should be considered that a fractionation of the biomolecules that
need high ligand density in order to be partitioned to a polymeric
phase usually requires the chemical activation of the DEX phase
3
via the tresyl–chloride method [23]. This is possible due to the
increased number of active hydroxyl groups, in comparison with
PEG, present in DEX that can be chemically activated with affinity
ligands.
The benzoate activation protocol exhibits interesting advan-
tages over the aforementioned activation techniques. In terms of
overall processing time, it requires only 5 h to generate an active
matrix. In addition, an important advantage is that the active ben-
zoyl groups in the polymer matrix may act as affinity ligands
for molecules with tryptophan residues. This implies integration
between the polymer activation technique and the ligand coupling
requirements. Overall, the benzoate activation protocol seems well
suited for recovery and purification of proteins in ATPAP systems
considering these advantages.
Even though the methodologies presented in Table 1 are tradi-
tional and represent several of the first approaches considered for
polymer activation, more protocols have been developed in order to
increase affinity partitioning in liquid–liquid systems. Electrostatic
interactions have been well studied in order to develop chemical
modifications that enhance this behavior towards target molecules.
Activation with carboxylic groups (in presence of diglutaric acid)
and primary amino groups [5] has been described for recovery of
antibodies and proteins, even though it must be emphasized that
amino groups tend to increase the pH level and thus have a lower
efficiency in product partitioning. Also, polymer activation with
pyrimidine groups has been described [3], emphasizing that active
groups interact electrostatically with target molecules. Neverthe-
less, because of the nitrogen atom in the pyrimidine group, the net
charge of the target product must be carefully monitored in order
to maximize system efficiency [3].
Active phosphate groups can be coupled to PEG chains through
an activation protocol involving phosphoryl-chloride [18]. It has
been found that increasing concentrations of PEG–PO4 in an extrac-
tion system generates an increment of miscibility between system
constituents. Therefore, higher concentrations of polymer and salts
are required to form the biphasic system, which favors the partition
behavior of target molecules towards a particular phase. Additional
techniques and methodologies for the activation of PEG molecules
are described in the literature [24,25].
Since activation procedures often require long reaction times,
optimization studies oriented in reducing the activation time may
further increase the potential of ATPAP at lab and industrial scale.
Furthermore, the development of versatile modification reactions
such as the PEG–benzoate (Table 1) or PEG–diglutaric acid will
pose serious advantages in chemical modification strategies, since
the whole protocol (which includes polymer activation and ligand
coupling) would be integrated into one step.
3. Polymer–polymer affinity aqueous two-phase systems
Traditional polymer–polymer ATPS formed by PEG and DEX
have been exploited to recover high value molecules that justify
the cost of the chemical forming phases, particularly dextran. In
addition, affinity enhanced polymer–polymer systems provide a
wide scope for the recovery of biological products. In this type of
ATPAP the polymer–ligand complex added to the system mainly
partitions towards the phase rich on that specific polymer (e.g.
PEG–ligand molecules partition to the PEG rich phase). Examples
of purified molecules in these systems include myeloma proteins
[15], inner membranes [23], human cells [26], commercial enzymes
[21,27,28], nucleic acids [24,29] and monoclonal antibodies [3,5].
Due to the particular characteristics of polymer–polymer systems
the studies have primarily focused on the recovery and purification
of high molecular weight products. Table 2 shows several exam-
ples of products that have been recovered using polymer–polymer
ATPAP.
 4
Table 1
Most common chemical modifications employed in ATPAP systems for biomolecule recovery and purification.

Polymer functional group Activation group Modified product Ligand requirement Ligand examples Reference

Yes – active amine groups: Dinitroflurobenzene, aminoacids [15]

F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13

Nucleophilic coupling

PEG Yes – oxirane active groups subject to Peptides, e.g., Glutathione, proteins, [19,20]
nucleophilic attack e.g., protein A, BSA

PEG Yes – active periodate groups: BSA, glutathione, carbohydrate binding [20]
nucleophilic substitutions modules

PEG No – active benzoyl groups allow Does not requiere biospecific ligand [18]
interaction with triptophan residues

PEG dextran Yes – Cl and O activated groups serve Proteins (avidin), coenzynes, cofactors [17,21–23,26,62]
as nucleophilic addition targets like ATP
 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13 5

Fig. 1. (A) ATPAP systems with affinity ligand (AL) chemically coupled to phase forming components. (A1) Polymer–salt ATPAP system with AL coupled in top phase polymer.
(A2) Polymer–polymer ATPAP system with AL coupled in bottom phase forming component. (B) ATPAP systems with AL free in solution in top or bottom phase. (B1)
Polymer–salt system with AL free in solution in top phase and polymer–polymer system (B2) with AL free in solution within bottom phase component. It should be noted
that the polymer solutions utilized can include traditional phase forming components such as PEG and dextran, but novel natural polymers can also be employed in each of
the ATPAP systems proposed.

Table 2
Selected examples of polymer–polymer aqueous two-phase affinity partitioning systems for recovery and purification of bioproducts.

Product System Modification Recovery yield Reference

IgG (human) Dextran T500/PEG 3350 PEG-benzyl 88% recovery/90% purity [3]
Human antibodies Dextran T500/PEG 3350 PEG-diglutaric acid 93% recovery yield [5]
IgG (human) Dextran T500/PEG 1450 PEG-IgG-HRPa 91% recovery/88% purity [11]
S-23 myeloma proteins Dextran T500/PEG 6000 PEG-dinitrophenyl Kp increase: 2.8–7 [15]
Thaumatin Dextran T500/PEG8000 PEG-gluthatione Kp increase: 0.27–4.6 [20]
Trypsin Dextran T500/PEG 8000 PEG-trypsin inhibitor Kp increase: 0.5–16 [20]
Biotinylated liposomes Dextran T500/PEG 3350 Dextran-avidin 95% recovery in BPb [22]
Biotinylated liposomes Dextran 2000/PEG 3350 Dextran-NeutrAvidin 94% recovery in BP [23]
Biologic membranes Dextran 40/PEG 3350 Dextran-NeutrAvidin 88% recovery in BP [23]
Red blood cells Dextran T500/PEG 6000 PEG-MPEG-IDA(II) and Dextran 95% recovery in BP [26]
Lymphoma cells Dextran T500/PEG 6000 Sephrose-IDA-Cu(II) 73% recovery in BP [26]
Plasmid DNA Dextran 40/PEG 600 GSTc + finger Kp increase: 0.005–29.42 [29]
␤-Galactosidase Dextran T500/PEG 8000 Dextran-benzoyl Kp decrease: 0.036–0.018 [65]
Lysozyme Dextran T500/PEG 8000 Dextran-benzoyl Kp increase from 0.20 to 0.85 [65]
␤-Galactosidase Dextran T500/Valeryl Dextran T500 Dextran benzoyl Kp increase from 0.046 to 0.18 [65]
Lysozyme Dextran T500/Valeryl Dextran T500 Dextran-benzoyl Kp decrease from 0.69 to 0.45 [65]
Lacrimal gland plasma membranes Dextran T500/PEG 3350 Dextran-WGAd 90% recovery in BP [66]
Escherichia coli inner membranes Dextran T40/PEG 3350 Ni-NTA resin in Dextran phase 93% recovery in BP [67]
a
HRP, Horseradish Peroxidase.
b
BP, bottom phase.
c
GST, Glutathione-S-Transferase.
d
WGA, wheat germ agglutinin.
6 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
Thaumatin and trypsin were purified by ATPAP systems formed
using PEG 8000 and DEX T500 [20]. Epichlorohydrin and Epoxy-
oxirane activation methods allowed coupling of glutathione and
trypsin inhibitor as biospecific ligands in PEG molecules. Thau-
matin, a flavor enhancer and sweetener protein, presented a 17-fold
increase in Kp (from 0.27 to 4.6) in modified ATPS systems with
PEG–glutathione in top phase. Trypsin, a proteolytic enzyme (EC
3.4.21.4), experienced a dramatic shift in partition coefficient from
0.5 to 16 (32-fold increase), in aqueous systems with trypsin
inhibitor bound to PEG. These results demonstrated that the recov-
ery and purification of the selected proteins could be dramatically
increased by using modified ATPS with biospecific ligands attached
to PEG molecules that partitioned to the PEG rich phase without
significant changes in the system composition [20]. In addition, the
authors established that the DS, which considers a saturation effect
in the system when a molecule and an affinity ligand are added in
excess to the system, could decrease system efficiency.
Partition of membranes and negatively charged liposomes with
a biotin spacer arm was studied in ATPAP systems with DEX 40/DEX
2000 and PEG 3350 [23]. Isolation of inner and outer membranes
represents an important area of opportunity for new drug delivery
strategies [23]. Membranes and liposomes were recovered in affin-
ity ATPS with deglycosylated avidin (NeutrAvidin) as a bioaffinity
ligand coupled (via tresyl chloride activation method) to dextran
molecules that partitioned to the bottom phase. Liposome parti-
tion was first studied in order to determine optimal parameters
for further purification of biotinylated membrane fractions. In sys-
tems with absence of NeutrAvidin, 90% of the liposomes partitioned
to the PEG rich phase. Addition of NeutrAvidin–DEX induced a
notable shift of biotinylated liposomes to the dextran rich phase,
from 85% in PEG rich phase (without ligand) to 90% in the dextran
bottom phase. The authors concluded that the interaction between
the biotin spacer arm and NeutrAvidin in liposomes and mem-
branes determines the partitioning behavior of these molecules.
This biological interaction represents a valuable approach for the
purification of biotinylated products.
Affinity partitioning of pre-purified plasmid DNA (pDNA) using a
PEGylated zinc finger-GST (Glutathione-S-Transferase) fusion pro-
tein as affinity ligand was studied in PEG–DEX ATPAP by Barbosa
et al. [29]. Purified pDNA based therapies emphasize the impor-
tance of scalable and cost effective process development. The
coupling of a fusion protein to a polymeric matrix reduces steric
hindrance commonly present in resin-coupled proteins [29]. PEG
600–DEX 40 and PEG 1000–DEX 500 systems were used for pDNA
isolation. Comparative experiments determined that when the
fusion protein was first administered in the system in its unPEGy-
lated form, its partition coefficient was of 37.89 and the partition of
the plasmid added to the system was calculated as 0.005. Neverthe-
less, pDNA partition coefficient increased 5900-fold (from Kp 0.005
to 29.42) when adding the PEGylated form of the proposed affinity
ligand to system PEG 1000–DEX 500. The authors confirm that a
strong interaction between the PEGylated fusion protein and the
pDNA allows the complex to concentrate in a specific phase (PEG
rich phase). PEG 600–DEX 40 systems presented a lower increase
in Kp (from 0.0003 to 2.42). However, when increasing the PEGy-
lated fusion ligand concentration to 293 ␮g/ml, pDNA partitioned
completely (100%) towards the PEG rich phase, thus representing
an efficient strategy for its recovery. The authors demonstrated the
potential of affinity partitioning of pDNA in ATPAP, emphasizing the
effect of polymer characteristics such as concentration and molecu-
lar weight, on ligand distribution through the system, which can be
exploited to develop new concentration and purification strategies.
Purification strategies using free ligands in solution have been
implemented in ATPAP to recover biological products. The princi-
pal advantage of this methodology is that no polymer activation
and ligand coupling are involved, reducing time and resource
consumption. Recombinant proteins tagged with a choline binding
module (C-LytA) were isolated by Maestro et al. [6] using ATPAP
with free choline as a natural bioligand. The C-LytA module con-
sists of six conserved ␤-hairpins that include four choline-binding
sites [6]. Hence, the affinity of C-LytA for choline groups and analog
compounds was used by the authors to design ATPAP strategies. A
strong interaction of the choline-binding sites present in the C-LytA
module with PEG was discovered. In consequence, proteins tagged
with the C-LytA module would experience an initial shift to the top
phase. On the other hand, addition of choline to the system reversed
the interaction between the C-LytA module and PEG, redirecting the
target molecule to the PEG-poor (bottom) phase. Green Fluorescent
Protein (GFP) fused to C-LytA was studied as a proof of concept. A
shift in the partition coefficient for the fusion protein from 186
to 0.5 was observed, with an 80% total recovery of native GFP in
the bottom phase. The main conclusion described by the authors
was the affinity partition modulation by choline residues present
in C-LtyA. Simplicity, rapidness, effectiveness and scalability confer
versatility to the proposed strategy.
Polymer based systems represent an important alternative for
product recovery since both phases could be chemically active.
Furthermore, recovery and purity yields of protein products on
such systems are usually high (Table 2). Nevertheless, it is clear
that process efficiency in ATPAP could be optimized. According to
several researches, salt addition in polymer-based systems is capa-
ble of increasing recovery yields, since marked shifts in ionic force
can favor specific affinity interactions between target particles and
affinity ligands [3]. In this tenor, salt effects are not always consid-
ered when applying ATPAP extraction methodologies even though
it has been determined that particular salts may positively enhance
product partitioning [15,29]. Also, the effect of pH on partitioning
should be considered in order to increase ATPAP efficiency. In addi-
tion, it is important to consider that even though polymer activation
techniques allow ligand coupling in order to favor product partition
in aqueous systems, spacer arms between the polymer and ligand
tend to have different lengths depending on the employed activa-
tion procedure [20]. In general longer spacer arms favor affinity
partitioning since the probability of the interaction between prod-
uct and ligand increases [22,23]. In this tenor, exploiting the effect
of the spacer arm length represents an interesting option in order
to increase recovery yields in ATPAP [22].
One important strategy within ATPAP is that of free ligands in
solution. In order to avoid time-consuming activation protocols for
polymers, direct addition of affinity ligands in order to enhance
product partitioning has been presented [6,29]. Despite the fact that
polymer activation is not necessary, a proper study of ligand parti-
tioning must be undertaken in order to characterize its partitioning
behavior, and thus determine its true potential in affinity extraction
procedures. Modification of the proposed affinity ligands could be
carried out in order to manipulate their partition behavior towards
either phase of the system. One of the most promising techniques
that could be used to modify affinity ligands is PEGylation. PEGyla-
tion reactions have been well studied and characterized and allow
the coupling of a PEG moiety to a particular compound, in this
case an affinity ligand [29]. Because of the chemical nature of the
modified ligand, it has been demonstrated that partitioning behav-
iors toward PEG rich phases are greatly favored [29]. Considering
this methodology, PEGylation of proteins, antigens or dyes could
be developed in order to modify ligand behavior within ATPAP sys-
tems and thus design efficient unit operations for recovery of whole
cells or stem cells, nucleic acids and cellular fragments. It must
be emphasized that even though PEGylation has been widely used
for therapeutic applications, its relevance and implementation in
aqueous affinity partitioning has not been extensively addressed.
Hence, it is expected that novel applications of this technology
coupled to ATPAP strategies will be seen in the near future.
 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
Polymer–polymer systems offer great versatility within ATPAP
(Table 2). Phase components characteristics maximize biological
interactions which can be exploited in order to develop novel
downstream processing protocols. Nevertheless, the cost of the
chemical forming phases in these systems should always be con-
sidered, since PEG and particularly DEX may represent a significant
cost at industrial scale. However, this issue can be addressed by
polymer and ligand recycling, applying back-transfer phase strate-
gies in order to maximize raw material medium life.
4. Polymer–salt affinity aqueous two-phase systems
Traditional polymer–salt ATPS have been used for the recov-
ery and purification of high value products. However, affinity
enhancement has not been extensively addressed as in the case of
polymer–polymer systems. A reduced number of cases are found
for these systems, and applications seem to be focused in purifica-
tion of commercial enzymes (Table 3). One of the mayor drawbacks
of using salt components in affinity enhanced liquid–liquid extrac-
tion methodologies lies within the biological interaction of ionic
groups of certain molecules towards the salt forming phase. The use
of certain salts in ATPS has been proven to increase the partition of
target molecules below certain limits, but above critical concentra-
tions a decreased pattern of solubility for proteins and biological
products can be observed. Important shifts in the ionic force of
the solutions are experienced, reducing essential ionic interactions
between target molecules and proposed affinity ligands. Neverthe-
less, applications have been reported using this type of systems for
ATPAP [28,30,31].
As an example, penicillin acylase (PA) partitioning behavior
in ATPAP polymer–salt systems has been studied [18]. PA is an
important hydrolytic enzyme that is used in the direct synthesis
of semi-synthetic penicillins [30,31]. Four PEG derivatives were
synthesized: PEG-benzoate (PEG-bz), PEG-trimethylamine, PEG-
palmitate and PEG-phenylacetamide (PEG-paa). PA partition in
PEG-bz systems presented a 14-fold increase in Kp (60% total recov-
ery) with 21% PEG-bz of the total PEG mass. For the particular
case of PEG-paa systems, PA presented an enrichment factor of 9.4
and an 11.5-fold increase in Kp. PEG-bz and PEG-paa presented
high affinity towards PA due to the fact that the interaction energy
required for the coupling of PA with these components was lower
than the interaction energy between PA and its natural substrate.
This increased the biological affinity of the target enzyme towards
the activated PEG molecules present in the PEG-rich phase. The pro-
posed PEG derivatives significantly increased PA partition in ATPAP
systems and the primary interaction was attributed to the phenyl
ring and amide groups present in the proposed affinity ligands [18].
An additional example for the application of ATPAP using free
ligand addition is the partition of chitinases in systems formed
by PEG 6000, dipotassium hydrogen orthophosphate (K2 HPO4 )
and free chitosan in solution as affinity ligand [28]. Chitinases are
hydrolytic enzymes that act selectively in chitin [32]. ATPAP sys-
tems were prepared with 22% PEG (w/w), 10% K2 HPO4 (w/w) and
0.5% (w/w) of a chitosan solution. Chitinases extracts from Neu-
rospora crassa, cabbage (Brassica oleracea) and puffballs (Lycoperdon
umbrinum) were loaded to independent systems. Chitinase recov-
ered from N. crassa presented a 34-fold purification increase and
86% of total recovery in top phase. Meanwhile, extraction from
cabbage and puffballs exhibited a 20-fold and 38-fold purification
increase with 80% and 88% total activity recovery in the PEG rich
phase, respectively. The authors suggested that due to the high
concentration of chitinases, non-specific interactions between the
product and the affinity ligand were favored, lowering the over-
all binding capacity probably due to steric hindrance. This effect
should be considered in order to develop efficient recovery and
purification strategies.
7
The use of protein carriers in order to increase the potential
of ATPAP has been also documented by Hye-Mee et al. [11]. They
reported the use of Horseradish Peroxidase (HRP) as a carrier for
human IgG as an affinity ligand. Model mixtures with rabbit anti-
human IgG (target molecule) and goat anti-mouse IgG (impurity)
were employed in order to establish the potential of ATPAP for the
purification of the target molecule. ATPS systems involving 10%
w/w PEG 1450 and 15% w/w potassium phosphate were used. Rab-
bit anti-human IgG partitioned towards the salt-rich phase when
added with other contaminants in the system without affinity lig-
ands. On the other hand, when human IgG–HRP was added to the
system, the complex exhibited higher affinity towards the top PEG-
rich phase (Kp 1.65). As a result, a recovery of 78.2% of the target
molecule was achieved.
Purification of glucose oxidase and ␤-galactosidase from
Aspergillus niger and Kluyveromyces lactis fermentation broths was
reported using modified PEG–sodium sulfate systems [33]. In gen-
eral PEG–sodium sulfate systems are less used in comparison
with PEG–phosphate ATPS. In this particular case four PEGs were
synthetized: PEG-trimethylamine (TMAionic interaction ligand),
PEG-benzoate (pseudo specific ligand), PEG-Coomassie brilliant
blue G-250 (pseudo-specific ligand), and PEGpalmitate (hydropho-
bic ligand). PEG-Coomassie favored the partition of glucose oxidase
towards the bottom phase and the contaminants to the upper
phase. ␤-Galactosidase was best recovered in PEG–TMA systems
(80% recovery and 7-fold purification increase). TMA acts as a
positively charged ligand that repels positively induced-charged ␤-
galactosidase favoring its partition towards the bottom phase. The
authors emphasized the affinity attractions between active groups
in PEG and contaminant proteins, which in turn provides a new
strategy for purifying target molecules.
One of the principal advantages polymer–salt systems offer
for protein recovery is the reduced viscosity and density of the
aqueous formed phases. That will result beneficial in the poten-
tial implementation at commercial levels [18,20]. However, further
characterization of hydrophobic, electrostatic and biospecific inter-
actions is needed, since PEG molecules tend to suffer important
modifications when affinity ligands are coupled to their organic
backbone. Addition of proteins or chemical compounds may
increase the hydrophobic character of the polymeric phase which
could enhance partition of bioparticles with hydrophobic domains
[18]. This is an important strategy when partitioning of molecules
with known hydrophobic domains will be developed, since recov-
ery yields could be positively improved.
For enzyme recovery, polymer–salt systems present several
points to be characterized in order to design proper extraction pro-
tocols. First, the use of high ligand concentration or high amounts
of modified polymer molecules has demonstrated to be an effi-
cient strategy to increase product recovery (thaumatin [20] and
glucoamylase [21]). Nevertheless, in these cases when excessive
concentrations of modified PEG were added, an important shift
of contaminant proteins was seen, thus reducing the overall effi-
ciency of the proposed systems. Since this problem is common
when designing ATPAP methodologies [15,18,34], response surface
analysis (RSA) or statistical methods could be employed in order
to develop prediction models and thus facilitate ATPAP design.
Another important aspect in affinity systems design lies within
optimum pH determination. Purification strategies for enzymes
tend to use natural substrates (as presented by Teotia et al. [28] in
chitinase recovery), in order to maximize product recovery yields.
In this tenor, the pH is the most important factor in the system since
enzyme purification must be carried out at a pH in which no enzy-
matic activity will be present and thus avoid degrading the affinity
ligand [28,34,35]. Once partition has been achieved, the enzyme-
rich phase can be transferred to a fresh salt phase and elute the
product of interest with addition of salts. In addition, pH levels
8 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
Table 3
Examples of polymer–salt aqueous two phase partitioning systems for recovery and purification of biomolecules.
Product System Modification
Thaumatin PEG 8000/phosphate PEG-gluthatione
Glucoamylase PEG 300/phosphate Starch as free ligand
Chitinase PEG 6000/phosphate Chitosan as free ligand
Penicillin acylase PEG 4000/phosphate PEG-benzoate
Penicillin acylase PEG 4000/phosphate PEG-trimethylamine
Penicillin acylase PEG 4000/phosphate PEG-palmitate
Penicillin acylase PEG 4000/phosphate PEG-phenylacetamide
Cutinase PEG 1500/phosphate Butyrate as free ligand
Phospholipase D PEG 6000-phosphate Alginate as free ligand
have a direct effect in biological interactions between natural sub-
strates and affinity ligands. Electrostatic bonding between target
molecules and proposed ligands heavily depends on pH level since
active groups in each molecule possess different pKa or isoelec-
tric point (pI) values. Considering that every molecule has different
active groups, the pH level will pose an important priority since
it will be the moderator of electrostatic interactions between tar-
get products and affinity ligands. Nevertheless, it must be also
considered that pH variations around the pI enhance hydrophobic
interactions while decreasing electrostatic forces.
An important option that has been developed in polymer–salt
systems consists in the coupling of molecules carriers to affin-
ity ligands in order to maximize product recovery in specific
phases [11]. However, the partition of the carrier–ligand com-
plex must be first characterized in order to propose a possible
extraction procedure, which could suppose a disadvantage of
employing this type of selective strategy. As can be concluded
when comparing data from Tables 2 and 3, recovery yields for
proteins are clearly lower in polymer–salt systems (particularly
those systems in which polymer components have been chemi-
cally modified to enhance partition). These partitioning behaviors
could be principally attributed to salt interference and ionic force
shifts. Salt-forming components interfere with the ionic nature of
active groups in affinity ligands and biological molecules, as a result
the implementation of ATPAP methodologies in polymer–salt sys-
tems is more limited since reduced affinity interactions could
be maximized within these extraction systems. In polymer–salt
systems, the direct competition between affinity ligands, their
binding capacity and cross-reactivity with molecules present in
the environment need to be properly characterized in order to
avoid undesired interactions. Since a direct economic comparison
between polymer–polymer systems and polymers–salt systems
will favor the latter, the recovery of proteins using polymer–salt
ATPAP results interesting despite the aforementioned limitation.
An important area of interest in which polymer–salt systems
could prove to enhance product recovery is that of anti-freeze
proteins (AFPs). These molecules play a vital role in certain
organisms in order to allow them to survive at subzero temper-
atures since they decrease the freeze point of the body fluids
non-colligatively [36–38]. Nevertheless, actual downstream pro-
cessing strategies seem to focus on chromatographic techniques
based on metallic affinity chromatography [39], size exclusion
[40] and hydrophobic interaction chromatography [41]. In addi-
tion, non-chromatographic techniques have been poorly addressed
and ATPAP protocols could be designed in order to purify these
bioproducts. Since these proteins exhibit affinity to ice molecules
through hydrogen bonds, Van der Waals and hydrophobic interac-
tions, ATPAP strategies could be developed using PEG derivatives
as affinity ligands such as PEG-benzoate, PEG trimethylamine, PEG
palmitate or PEG phenylacetamide. In this same tenor, molecules
such as phycobiliproteins (B-Phycoerythrin, C-Phycocyanin) which
Recovery yield Reference
Kp increase from 0.21 to 0.71 [20]
Kp decrease from 6.6 to 0.73 [21]
90% recovery in UP after final elution [28]
14-fold increase of Kp (0.1–1.4) and 60% recovery in a single [18]
step extraction
Kp of 2.1 with 70% recovery in single step extraction [18]
Kp of 3.22 and 0.56 for contaminant proteins; 75% recovery in [18]
single step extraction
Kp of 2.3 and 0.1 for contaminant proteins; 69% recovery in [18]
single step extraction
98% recovery in first extraction and Kp of 135 [34]
85% recovery in UP and 78-fold purification factor [35]
are considered high added value molecules [4] could be recovered
within this affinity approach. This represents an important research
area for polymer–salt ATPAP systems, since a low number of stud-
ies with non-chromatographic techniques have been implemented
for AFPs and phycobiliproteins.
5. Affinity enhanced ATPAP systems using alternative
phase components
Affinity enhanced liquid–liquid extraction systems have been
developed with phase components different to PEG, DEX or tradi-
tional salts. The use of surfactants and natural polymers has been
previously reported because of their versatility in terms of ATPAP
systems development. In addition, natural components exhibit
important advantages over traditional polymers since they are
considered environmental friendly reagents. Purification of com-
mercial enzymes [42], fusion peptides [43] and cellular membranes
[44] has been reported (Table 4) using alternative materials in
ATPAP systems.
Aqueous two phase micellar systems have proven to work as an
alternative to traditional ATPS. These systems are formed when cer-
tain surfactants (ionic, zwitterionic and non ionic) are mixed with
an aqueous or organic phase above a critical micelle concentration
(CMC) [45]. When aqueous phases are employed, the hydropho-
bic tails of the surfactant molecules tend to aggregate in a circular
manner, forming structures known as micelles. On the other hand,
when organic components are used, the hydrophilic heads form
aggregates with the hydrophobic tails positioned in the exterior in
a structure know as reverse micelle. Surfactants used for micelle
formation can be modified with affinity ligands in order to increase
partition of interest molecules [46]. Therefore, micellar ATPAP pose
an interesting alternative to traditional ATPS.
CBM9-Green Fluorescent Protein (CBM9-GFP) was partitioned
in a micellar system with n-decyl-␤-d-glucopyranoside (C10 G1 ) as
phase former surfactant and affinity ligand [43]. Since C10 G1 acts as
phase forming component and ligand, the proposed binary system
(water/surfactant) significantly simplifies the recovery and purifi-
cation of the product of interest. Affinity partitioning of CBM9-GFP
was studied first with the presence of glucose, in order to demon-
strate that this carbohydrate acted as a potential inhibitor in the
system. At a 55 mM glucose concentration, the Kp for CBM9-
GFP was 0.473 ± 0.005, while in systems without glucose a Kp of
3.1 ± 0.2 was achieved. The observed partition coefficients show
the strong interaction between the CBM tag and the surfactant. The
use of alternative polymers to PEG and DEX is an important area
of opportunity for ATPAP systems. Different polymers have been
proposed to replace PEG in ATPS extraction systems [15,34,42,47]
in order to construct more economic strategies. Several research
groups have proposed alternative polymers that may act as replace-
ments of the traditional polymeric components. The development
of thermoseparable copolymers made of ethylene oxide and
 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
Table 4
Examples of aqueous two phase affinity partitioning systems using non-traditional polymers and phase forming components.
Product System
S-23 Myeloma Proteins PEO 6000/Dextran T500
␤-Mannanase Guar Gum-PES 200
CBM-9-GFP n-Decyl-␤-d-
glucopyranoside/water
Membrane proteins Tween 20/Dextran 500
Glucose-6-phosphate dehydrogenase PEG 6000/PES 100
Cutinase with peptide fusions BREOX PA G (EOPO) 1000-Reppal
PES 200
Cellulase PEO-dextran T500
a
FL, free ligand.
propylene oxide (EOPO polymers) constitutes an interesting alter-
native for ATPAP systems. In addition, EOPO components are
commonly mixed with natural starch polymers to form biphasic
extraction systems [34]. It has been determined that affinity ligands
can be attached to either phase forming molecules (EOPO or natu-
ral polymer phase), thus generating an ATPAP system. Also, natural
based components tend to be compatible with a wide variety of
natural substrates, which increases the possibilities of developing a
novel ATPS based procedure [42]. Despite these advantages, the use
of alternative phase components is not extensively characterized.
In this context, the phase formation diagrams or binodal curves
must be constructed in order to validate the experimental designs
and further purification protocols.
Cutinase selective partitioning was studied in modified ATPS
conformed by polyalkylene glycol 50 A 1000 as top phase form-
ing polymer and hydroxypropyl starch (BREOX-PES 200) as bottom
phase polymer [47]. A dual affinity method was applied in which
fusion tags (WP = Trp-Pro)2 , (WP)4 and Thr-Gly-Gly-Ser-Gly-Gly-
(WP)4 were attached to the enzyme and served as receptors for
specific detergents. It was first determined that fusion tags did not
affect significantly cutinase partition in the aqueous systems. Con-
sidering this, addition of non-ionic detergents to the systems in
order to increase the fusion tag affinity was investigated. Tween
20, Tween 80 and Triton X-100 were added (1% w/w) obtaining Kp
fold increases of 1.84, 1.55 and 6.4, respectively. However, addi-
tion of a 2% w/w solution of pentaethylene glycol monolauryl ether
(C12 E5 ) to the Breox-PES 200 systems allowed a 6.6-fold increase
in Kp and 5.8-fold increase in purity compared to systems without
affinity ligand. A tag effect (defined as the ratio between partition
coefficients for the fusion protein and the native type protein) of 14
for the TGGSGG-(WP)4 tag was observed, indicating that this deter-
gent was more efficient than the other studied to increase selective
partition of cutinase. The authors concluded that depending on
the fusion tag employed the interaction between a molecule and
a matrix can be increased. Furthermore, the addition of non-ionic
detergents can modify selectively the partition of fusion proteins
in order to increase their partition to one of the phases in an ATPS.
In addition, some characteristics of the employed polymers should
be emphasized. Even though PES 200 can be found commercially
at lower economical costs than dextran, the relative time stock
solutions are functional may be limited due to its biodegradability
potential [48]. Therefore, adequate storage and processing strate-
gies should be used in order to avoid unwanted degradation of the
polymer.
Carbohydrate binding modules (CBM) are natural tags present
in enzymes that facilitate substrate targeting and coupling [42].
Antov et al. [42] employed the renewable polysaccharide guar
gum galactomannan and starch in a novel ATPAP system to purify
␤-mannanase from Cellulomonas fimi. This enzyme has a CBM
with high specificity towards mannans. The approach was to
9
Modification Recovery yield Reference
Dinitrophenyl-PEO 2.5-fold increase in Kp (2.8–7) with [15]
presence of modified PEO
Galactomannan as FLa Kp increase from 1.37 to 7.06 [42]
C10 G1 (simultaneous phase 6-fold increase in Kp from 0.4 to 3.3 [43]
former and affinity ligand)
Dextran-cobalt affinity resin 75% recovery in detergent phase [44]
Cibacron Blue F3GA (FL) ATPS with dye induced an increase in [60]
Kp from 0.73 to 1.59
Triton X-100 and C12 E5 as FL’s Kp increase from 0.89 to 10.35 of [61]
recombinant cutinase with fusion tags
Maleic acid-PEO 2-Fold increase in Kp for cellulose with [68]
40% modification rate at PEO phase
exploit the affinity of the protein binding module to the galac-
tomannan polymer in the aqueous extraction system. The natural
polymers hydroxypropyl starch PES 200 (Reppal) and Guar gum-
galactomannan (9% (w/w) and 3.6% (w/w) respectively) were used
as phase forming components. This system allowed a top phase
(guar gum-galactomannan) recovery yield of 90% with a Kp of 7.06
for ␤-mannanase. With the obtained results, it can be noted that
diverse CBM could be employed for protein purification. Hence,
a general extraction procedure for proteins could be developed if
each target molecule is fused with a specific binding module. This
would represent an important advantage in the development of
novel downstream processing strategies.
Affinity partitioning of human immunoglobulin G (IgG) from
Chinese hamster ovary cells has been also studied on ATPAP
systems constructed with non-conventional constituents [49].
Purification was achieved by employing a system with 8% w/w
copolymer of ethylene oxide/propylene oxide (UCON), 6% w/w of
DEX 500 and triethylene glycol–diglutaric acid as affinity ligand
(TEG–COOH). Addition of TEG–COOH generated an improvement
on both extraction yield and purity of human IgG in the top
phase. The significant increase in log Kp (from −0.2 to 3.5) of IgG
was attributed to electrostatic interactions between the positively
charged IgG and the negative carboxylic groups of TEG-COOH [49].
According to surface response models generated by the authors, the
optimal ATPS for the purification of IgG was determined to contain
10% w/w UCON, 5.5% w/w dextran and 20% w/w TEG–COOH. In this
particular case, the use of optimization methodologies generates
valuable information for downstream processing strategies.
One of the current trends within natural occurring polymers
is their employment as phase forming components and affinity
ligands in a simultaneous manner [42]. According to liquid affin-
ity partitioning theory, a complex number of variables have to
be determined in order to design an ATPAP extraction procedure,
one of the most important being the presence of a specific affinity
ligand. If ligands are to be coupled or free in solution, their inclu-
sion to the system requires a characterization procedure in order
to determine optimum concentrations of phase forming compo-
nents, ligands and sample load. In this tenor, if a proposed natural
polymer can also serve as a specific ligand for product recovery,
this could reduce ATPAP complexity since no additional charac-
terization should be made, and the system could be considered a
traditional ATPS although the presence of this affinity component
would enhance product recovery.
Purification of ␤-mannanase in a Guar-gum galactomannan/PES
starch system was possible since the upper phase of the system
was the natural substrate for the target enzyme [42]. In addition
to the above mentioned strategy, the authors also concluded that
the pH of the system had a significant effect on the enzyme since
the product should be in a non-catalytic form. Even though target
products can be recovered and further eluted in a fresh salt phase
10 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
(S-23 myeloma proteins [15] or ␤-Mannanase [42]) product stabil-
ity could be affected if an adequate system pH is not used, favoring
the partition of contaminant molecules towards the target phase.
A disadvantage of natural polymers is their high viscosities.
Despite the fact that natural polymers (Reppal PES starch or Guar
Gum Galactomannans), are considered environmental-friendly,
high viscosities must be reduced in order to employ them as phase
forming chemicals. Pretreatments based on heat and partial enzy-
matic hydrolysis have been proposed in order to fulfill this objective
[42]. Nevertheless the cost-effective approach within the natural
system would be hampered. As with polymer–polymer systems,
the use of modified natural polymers in high concentrations may
enhance protein recovery since a biocompatible environment will
be available for the product of interest [15,42]. However, it should
also be taken into account that once the stoichiometric limits
for ligand concentration and polymer addition are reached, sys-
tem saturation is achieved. In terms of affinity systems composed
with alternative phase components and the present discussion and
examples described within this section and Table 4, protein recov-
ery is well suited with this technology. Starch based polymers and
other plant polysaccharides (e.g. guar gum) allow protein recovery
once they have been pretreated. Furthermore, their natural prece-
dence gives them added value since actual global trends emphasize
the importance of working with natural resources [34,42,48]. Natu-
ral polymers are environmental-friendly components with biologic
characteristics suitable for their application in downstream pro-
cessing operations. However, the production of such materials in
some cases needs to be standardized, which represents an impor-
tant issue to be addressed for the future application of such phase
forming chemicals. Also important is the chemical stability of the
polymer solutions, since several natural polymers cannot be pre-
pared and kept in stock for extended time.
6. Trends and challenges of ATPAP
One of the mayor trends in downstream processing involves
process integration and intensification. In this context, the imple-
mentation of ATPAP systems could provide an integrative approach
in process design. Biological affinity strategies can be used, as seen,
to purify commercial enzymes [5,6,21], membranes [23], plasmidic
DNA [24], and recombinant proteins [6] achieving high recovery
yields. One of the mayor advantages of working with ATPAP sys-
tems is their relative ease of set up and construction, minimizing
chromatography based strategies.
Nevertheless, the well-established chromatographic techniques
applied for the selective recovery and purification of biological
products could possibly hinder ATPAP application at industrial lev-
els. The main comparison between these techniques involves their
efficiency in terms of yield and purity for a specific product, and the
economic aspects of each one. In terms of product yield and purity,
a vast number of publications can be found in terms of chromatog-
raphy or affinity chromatography for product purification.
Table 5 presents a selective comparison of representative
biomolecules studied with ATPAP and chromatographic tech-
niques. In terms of efficiency, it can be seen that recovery yields
for human IgG [3,50,51] and CD34+ stem cells [53,54] are very
similar since they tend to be above 90%. Even though the proto-
cols for antibody purification with affinity chromatography have
been well studied in the past decades, stem cell recovery through
cell sorting methodologies will be insufficient in order to meet
the need for future stem cell therapies due to their low scalabil-
ity potential [53]. Since recovery yields are comparable with those
obtained with MACS techniques, authors have emphasized the rela-
tive simplicity of ATPAP for recovery of stem cells and thus, provide
a more economical and scalable technology for future stem cell
bioprocesses [54]. Nevertheless, as presented in Table 5, actual
purity standards for stem cells have not been well addressed with
liquid–liquid fractionation techniques. The most recent applica-
tion of ATPAP for CD34+ stem cells recovery presented yields of
42% in purity. This purity is significantly lower than that obtained
with traditional cell sorting methodologies [53]. In this case, the
strategy presented by the authors involved a monoclonal antibody
against the CD34 surface antigen, which favored partition of tar-
get cells to the PEG rich phase of the constructed systems. Even
though a strong immunoaffinity interaction was established, a cen-
trifugation step was employed for target cell concentration in the
interphase of the system, which also concentrated an important
number of contaminant cells. In this tenor, affinity strategies based
on target product concentration in the top or bottom phase should
be established, in order to avoid working with interphase steps,
which tend to increase the presence of contaminant molecules. In
addition, it should be considered that the scalability, integration
and intensification potential of ATPAP for stem cell recovery are
comparative advantages against traditional cell sorting method-
ologies (FACS and MACS), which cannot be employed at industrial
levels [53]. Although ATPAP methodologies for stem cell purifica-
tion have not addressed purity standards, it must be emphasized
that overall recovery yields with this technique are superior to cell
sorting methodologies. As reported by Sousa et al. [54], recover-
ies of 95–96% of CD34+ stem cells have been achieved by liquid
affinity partitioning and just 85% recovery has been reported with
MACS procedures. In this context, improved ATPAP will result in an
increase in purity for stem cells. Consequently, this extraction tech-
nology could prove to be one of the most important at industrial
level, since a critical advantage relies on its potential for bioprocess
scale-up.
In terms of enzyme recovery, penicillin acylase was selected
since recovery through ATPAP was lower when compared to
metallic affinity chromatography (IMAC). It was determined that
recovery and purity could be maximized with pre-established chro-
matographic methods and by optimization of the affinity resins in
order to increase superficial area and decrease pressure drops, and
thus confer an increased scalability potential [52]. However, it is
important to consider that the recovery of the enzyme with ATPAP
offers advantages of simplicity, scale up and potential of process
intensification [18].
As it has been discussed, recovery yields and purification
achieved using ATPAP and chromatographic methodologies are
similar. Even though ATPAP has not been extensively characterized,
its scale up potential and intensification capabilities could posi-
tion this alternative technique as a valuable approach in industrial
applications. Furthermore, the current existing process challenge
that the industry is facing will most likely require the use of com-
bined chromatography and non-chromatography approaches. For
the particular case of penicillin acylase recovery, liquid affinity
partitioning can provide similar results when compared to stan-
dard affinity chromatographic-based protocols (see Tables 2 and 3).
Nevertheless, it must be considered that the effect of several param-
eters must be characterized in order to obtain optimal results.
One of the principal strategies to increase soluble protein recovery
and purity relies on the strict control of electrostatic interactions
between amino acid residues and active sites present in affin-
ity ligands. This can be manipulated by studying the optimum
pH levels for protein stability and activity within an extraction
system, since different behaviors are expected for each protein
[20,28]. An additional strategy to increase product partitioning
is the implementation of affinity ligands that exhibit hydropho-
bic domains that will enhance recovery of molecules with similar
domains or structures [18]. It should be noted that this effect can
be maximized when protein extraction is made near the isoelec-
tric point, since product solubility will be low and thus facilitate
 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
Table 5
ATPAP and chromatographic techniques applied in bioproduct recovery.
Product Application Method
Human IgG Therapeutic treatments ATPAP
Protein A-Affinity
chromatography
Penicillin Synthesis of ATPAP
Acylase semi-synthetic IMAC chromatography
penicillins
CD34+ Therapeutic treatment ATPAP
stem cells of selected dystrophies
MACS device
hydrophobic interactions. In addition to the previous technical
comparison between ATPAP and chromatographic methodologies,
the bioprocessing trends and economic aspects of these two tech-
niques should also be considered in order to establish the appealing
of ATPAP for its future extensive use on the biotechnology indus-
try. According to the potential trends in bioprocess engineering
and monoclonal antibodies production, current advances in pro-
duction systems will condition the actual downstream processing
strategies to process higher concentrations of target molecules
[55–57]. In this context, several promising alternatives, like aque-
ous two phase affinity partitioning, could emerge in order to reduce
the number of unit operations in a process and at the same time
enhance the overall productivity when coupled to a smaller affinity
chromatographic step [55]. Rosa et al. recently published a whole
economical comparison between aqueous partitioning and affin-
ity chromatography for the selective recovery of high added value
bioproducts [55]. In the proposed investigation, a pharmaceutical
plant was designed and modeled with mathematical and economi-
cal software in order to predict total production cost of 1.2 tons of a
mAb per year. Two processes were designed: (1) one with a down-
stream process based on ATPS and (2) another that included affinity
chromatography. Total inversion costs were of $10,959,672 USD
for the ATPS process against $ 12,695,523 USD for the chromato-
graphic approach. In terms of annual operating costs, ATPS included
$ 8.7 million dollars and affinity chromatography supposed $ 14.4
million dollars. The most expensive component of the operating
costs was the affinity resin employed in chromatographic proce-
dures for mAb extraction ($ 16,250 USD/L of resin which constitutes
79% of the operating cost). On the other side, raw materials consti-
tute the 58% of the total costs for the ATPS annual expenditures
(PEG and salts as phase forming components). The authors found
that working with low titers of mAb (1–2 g/L) decreased annual
operating costs in chromatography but increased ATPS expendi-
tures. The opposite behavior was found when increasing the mAb
concentration to 4–7 g/L, since ATPS can process more sample of
the desired product without changing the system composition. It
was concluded that the proposed ATPS methodology could contin-
uously process the same amount of mAbs than a chromatographic
based process, with reduced operating costs and high titer process-
ing capabilities [55]. Hence, industrial processes with ATPS could
represent an alternative to affinity chromatography or traditional
chromatography in order to reduce operating costs and facilitate
process intensification. The biocompatibility and low toxicity of
renewable polymers make them attractive constituents of ATPS
related techniques. Currently, there is a clear interest for studying
the use of new polymers such as natural occurring polysaccharides,
like starch. The use of guar gum, a non-ionic storage polysaccha-
ride from guar plants, has become a primary focus of study because
of its neutral chemistry and biological activity when used in aque-
ous two-phase systems [42]. Hydroxypropyl starch and guar gum
11
Conditions Yield Reference
Dextran T500-PEG 3350 (PEG benzyl) 91% recovery and > 90% purity [3]
Product elution with 0.1 M glycine HCl 95% recovery and > 93% purity [51]
pH 3 and column regeneration 50 mM
NaOh
PEG-phosphate (PEG trimethylamine) >70% recovery [18]
Protein capture with pH 8.5 and 0.5 M 92% recovery and >90% purity [52]
NaCl, elution with 1 M NH4 Cl buffer
with 10 mM sodium phosphate
PEG 8000-dextran T500 (with anti 95.5% recovery with 42% purity [54]
CD34+ mAb)
Immunomagnetic cell sorting from a 85% recovery and >95% purity [53]
50% SSEA-1+ mESC cell mixture
polysaccharides are readily being used for their phase forming
capabilities and their user-friendly disposability.
The nature and origin of affinity ligands used on ATPAP has
experienced significant changes over time. First reports on ATPAP
systems emphasized the specific interactions between affinity lig-
ands and molecules of interest. Such ligands were chemically
designed and synthesized to exert specific affinity. During the last
decade the transition from chemical molecules to ligands such
as enzymes, carbohydrate binding modules (CBM’s), biologic sub-
strates and antigens, has become an important trend in ATPAP
applications. This fact marks a distinction between first generation
and second generation ATPAP systems. It can be expected that a
great variety of biomolecules will be studied within the next few
years as possible bioligands.
In terms of free affinity ligands in solution, they provide several
advantages over chemically coupled ligands. Polymer activation
techniques are time and resource consuming, while free ligands do
not need to be covalently coupled to the phase constituent, hence
time saving protocols can be developed. Nevertheless, after placing
an affinity ligand in the system, the molecule by itself must exhibit
a strong preference for either the top or bottom phase in order to
develop a purification strategy based on that behavior. In addition,
the effect of adding a particular ligand should be well studied. Ide-
ally it would be expected that the system characteristics such as VR
and composition stay well balanced and unaltered.
Affinity partitioning has studied a wide scope of biomolecules
in liquid–liquid extraction systems and the primary focus of this
technique has been non-therapeutic proteins [33,58–62]. How-
ever, the application of ATPAP for the commercial recovery of high
commercial value biologicals with therapeutic uses is expected.
One particular example makes reference to the recent advances
on monoclonal antibodies (MABs) therapies [3,63]. In this con-
text, the use of ATPAP systems may be an attractive alternative
to chromatography-based strategies. Another area that expects a
sudden increase in demand is that of gene therapy mediated by
plasmidic DNA vaccination [24,29]. Based on the numerous on-
going trails for pDNA therapies novel strategies for the recovery
and purification of these macromolecules are going to be needed
in order to satisfy the increasing demands in this niche [24,29].
The effectiveness of ATPAP has been proved on a wide variety of
biologicals, primarily macromolecules, cell fragments and whole
cells. Fig. 2 shows the correlation among the absolute change in
ln Kp and average particle diameter (nm) of different types of bio-
logical products (specific characteristics of the products included in
Fig. 2 are shown in Table 6) when ATPAP strategies are used instead
of traditional ATPS. Most of the proteins that have been studied on
ATPAP have a molecular weight lower than 100 kDa (besides anti-
bodies), while nucleic acids are between 100 and 125 kDa. Small
cells and cell fragments (primarily membranes) have been effec-
tively recovered in ATPAP systems constituted by two polymeric
12 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13

Fig. 2. Correlation of absolute change in ln Kp with the nature and average particle size of selected examples of macromolecules recovered in polymer–polymer and
polymer–salt ATPAP systems. Despite molecular nature and particle size, ATPAP methodologies can be widely adapted for recovery and purification of molecules in
bioprocesses.

Table 6
Selected examples of biomolecules partitioned in ATPAP systems according to increasing particle size.

Number System Ligand Product Size Reference

1 PEG 8000-phosphate Gluthatione Thaumatin 3.5 nm [71] [20]

2 PEG 4000–Phosphate Benzoate Penicillin Acylase 5.5 nm [18] [19]
3 PEG 10000–dextran 40 PEG–Cu-IDA (DSa ) pDNA 2686 bp [72] [24]
4 PEG 600–dextran 40 GST-zinc finger Protein (DS) pDNA 2693 bp [29] [29]
5 PEG 3350–dextran 500000 Glutaric acid (FL) IgG 28 nm [73] [3]
6 PEG 3350–dextran 500000 Diglutaric acid IgG 28 nm [73] [5]
7 PEG 3350–dextran 500000 Avidin Biotinylated Liposomes 45 nm [74] [22]
8 PEG 3350–dextran 2000 NeutrAvidin Microsomal membranes 150 nm [74] [23]
9 PEG 8000–dextran 500000 Anti-human IgG Erythrocytes 7700 nm [75] [70]
10 PEG 8000–dextran 500000 Anti-mouse IgG NS-1 Cells 13,000 nm [76] [69]

Examples presented in this table refer to the selected molecules from Fig. 2.
a
DS, double stranded.

phases. As can be observed in Fig. 2, for these selected biological
products the ln Kp values range from >1 to <9, demonstrating
that regardless identity, nature and size of the biological products
a specific partitioning can be achieved using ATPAP methodologies.
It would be of great interest to study the recovery and purification
potential of ATPAP for several other macromolecules of commercial
interest such as phytochemicals, pigments, hormones, antifreeze
proteins, phycobiliproteins, transcription factors, polysaccharides
and tandem/fusion proteins. In addition, recovery of bionanoparti-
cles such as dendrimers, organelles and virus-like particles would
suppose an interesting field for ATPAP partitioning. In this context,
research groups and biotechnological industries could apply these
new methodologies in order to design bioprocesses that involve
less unit operations [62–67], hence increasing purity and over-
all recovery yield. Furthermore, the recovery and purification of
low molecular weight compounds of commercial interest, such as
nutraceuticals and natural pigments on ATPAP may represent an
interesting new field to explore.
Regarding the isolation of the biomolecules of interest after
ATPAP, strategies for chemical or biological displacement of the tar-
get molecule from the affinity ligand should be further studied and
optimized. Desorption methodologies have been poorly addressed
or emphasized on the available scientific literature. Traditional
methodologies including ionic force shifting, pH adjustment, and
the addition of a displacer may be used to promote desorption of
the molecule of interest from the affinity ligand. It is expected that
efficient displacement methodologies will be developed and ATPAP
will gain increased popularity among downstream processing unit
operations.
7. Conclusions
Over the last two decades, affinity partitioning in liquid–liquid
extraction systems has experienced a marked growth. Since almost
every biological molecule exhibits a natural affinity towards a par-
ticular substance, general ATPAP protocols may be developed for
a wide variety of organic particles, which could possibly trigger
new research areas within downstream processing. One mayor
trend in ATPAP systems is using new raw materials as phase form-
ing components, emphasizing eco-friendly polymers. In addition,
development and implementation of new molecules as affinity
ligands that have not been used in affinity partitioning will sup-
pose an important area of opportunity in the near future. Major
challenges in ATPAP, such as the optimization of displacement
protocols, the characterization of novel and efficient chemical mod-
ification methodologies, and the cost of some affinity ligands, may
be addressed in the next few years. This could probably increase
 F. Ruiz-Ruiz et al. / J. Chromatogr. A 1244 (2012) 1–13
ATPAP popularity in the development of novel downstream strate-
gies for recovery and purification of biological products. Based on
the advantages described herein it can be concluded that ATPAP
may be an attractive alternative to traditional chromatographic
techniques at industrial scale.
Acknowledgments
The authors want to thank Tecnológico de Monterrey Research
chair (Grant CAT161) and CONACyT (the National Council for Sci-
ence and Technology in Mexico) for the scholarship #295368.
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