Process Biochemistry 39 (2004) 889896

Large-scale separation and production of engineered proteins,

designed for facilitated recovery in detergent-based aqueous
two-phase extraction systems
Klaus Selber
a
, Folke Tjerneld
b
, Anna Colln
b
, Teppo Hyyti
c
, Tiina Nakari-Setl
c
,
Michael Bailey
c
, Richard Fagerstrm
c
, John Kan
d
, Joop van der Laan
d
,
Merja Penttil
c
, Maria-Regina Kula
a,
a
Institute of Enzyme Technology, Heinrich-Heine University, Dsseldorf, D-52426 Juelich, Germany
b
Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, S-22100 Lund, Sweden
c
VTT Biotechnology and Food Research, P.O. Box 1500, FIN-02044 VTT, Espoo, Finland
d
Genencor International, B.V., Archimedesweg 30, 2333 CN Leiden, The Netherlands
Received 11 July 2002; received in revised form 7 April 2003; accepted 22 May 2003
Abstract
The feasibility and scalability of extraction in detergent-based aqueous two-phase systems for the separation of proteins from culture
broth is demonstrated. At the same time the large-scale production of a fusion protein and the inuence of cultivation scale on the efciency
of separation were investigated. An amphiphilic fusion protein EGIcore-HFBI was chosen, consisting of the catalytic core of the cellulase
endoglucanase I and the small protein hydrophobin I, expressed homologously in Trichoderma reesei. Using the technical nonionic detergent
Agrimul NRE 1205 the separation was successfully scaled up to 1200 l. No differences in yield or in partition coefcient were observed at 10
ml and 1200 l scale. Changes in the fermentation temperature and scale, however, can inuence the properties of the protein and thus alter
partition coefcient and yield. The decreased separation efciency appears to be correlated with changes in glycosylation at lower cultivation
temperatures.
2003 Published by Elsevier Ltd.
Keywords: Detergent-based aqueous two-phase systems; Large-scale extraction; Endoglucanase I; Hydrophobin fusion protein
1. Introduction
Extraction in aqueous two-phase systems (ATPS) is a
reliable method to clarify, purify and preconcentrate pro-
teins and other biomolecules [13]. Only PEG/salt systems
are reported to be in industrial use today. In such systems,
PEG and certain salts (phosphates, sulphates) are needed
in moderate concentrations to accomplish phase formation.
Economically, ATPS have certain advantages especially
with respect to costs of labour, energy and investment. One
drawback, however, is the relatively high costs of chemicals,
if no recycling is applied [4,5]. In the meantime, sewage
costs are increasing heavy and have to be considered. A
newer development in ATPS could possibly overcome these

Corresponding author.
E-mail address: mrk3372002@yahoo.de (M.-R. Kula).
drawbacks through the use of only one single-phase form-
ing component at lower concentrations. These so-called
detergent-based systems or cloud-point extraction systems
form two phases in solutions of nonionic detergents above
a certain temperature, referred to as the cloud-point tem-
perature. These systems are suitable for the separation of
hydrophobic and amphiphilic molecules.
Most applications of the detergent-based ATPS target
the solubilisation and separation of membrane bound sub-
stances. This has been done in a broad variety of laboratory-
scale experiments. Membrane proteins have been solubilised
from cells including plants and neural cells, archaebacteria,
eubacteria and yeasts, as summarized by Snchez-Ferrer
et al. [6] and Hinze and Pramauro [7].
Few large- or pilot-scale experiments and applications of
ATPS are reported in the literature. In 1982, Kroner et al.
[8] showed that scale up of extraction processes is easy and
0032-9592/$ see front matter 2003 Published by Elsevier Ltd.
doi:10.1016/S0032-9592(03)00198-5
890 K. Selber et al. / Process Biochemistry 39 (2004) 889896
predictable for PEG/salt systems. Kim et al. [9] report an
amylase extraction on a 50,000 l scale and Heinsohn et al.
[10] report a large-scale industrial chymosin process. These
and other processes are referenced in a review focussing on
the application of ATPS [3].
Concerning detergent-based systems, Minuth et al. [11]
report a 5 kg scale experiment isolating the membrane bound
cholesterol oxidase from Nocardia rhodochrous using a
technical detergent (C
1218
E
5
). In the present investigation,
a similar nonionic detergent Agrimul NRE 1205 was used
as phase forming polymer. It is a polyoxyethylene with a
carbon chain distribution consisting to a large extent of C12
chains and minor fractions of C14, C16, and C18, which is
derived from plant oil rich in C12 lipids. The cloud-point
(the point where the dehydration of the detergent and thus
phase separation occurs) at a concentration of 2% Agrimul
NRE 1205 in water is at 22

C. The phase diagram and the

physical properties of the detergent are discussed in detail
by Selber et al. [12].
Several recent studies to facilitate separation by geneti-
cal engineering of target proteins can be found in the liter-
ature. Investigations published before 1999 are summarized
in Berggren et al. [13]. These studies indicate that tagging
of proteins using small molecules can be a versatile tool,
which enables the directed partition in ATPS for the separa-
tion of a target protein. This has also been demonstrated by
additions of small peptide tags to the two-domain cellulase
endoglucanase [14]. However, difculties in the expression
of fusion proteins with hydrophobic peptide tags have been
observed [15].
The target protein chosen for this investigation was a ge-
netically engineered protein consisting of the fungal cel-
lulase endoglucanse I (EGI) and the amphiphilic protein
hydrophobin I (HFBI) from Trichoderma reesei. Since, a
part of EGI (the cellulase-binding domain) was replaced by
HFBI the protein is called EGIcore-HFBI. HFBI is a class
II hydrophobin with a molecular mass of 7 kDa. The fusion
protein is expressed homologously under the control of the
strong cbh1 promoter.
An important application of cellulases is the treatment
of cotton fabric, e.g. for the production of stone-washed
jeans or in the wash. Cellulases are bulk products, which
are usually applied as cocktails without purication after
the clarication and concentration of the culture broth. In
order to be able to prot from a specic application of a
single cellulase a separation procedure must be inexpensive
and applicable on a large-scale. The detergent employed
for phase separation in detergent-based systems could in
principle be removed from the protein and recycled [16].
However, in some applications the detergent may provide a
perfect formulation and does not need to be removed.
The goal of this work was to examine the technical
feasibility of large-scale purication of enzymes using
detergent-based ATPS. For this aim a direct scale up from
10 ml to 1200 l was done in the downstream processing
while the cultivation was scaled up from 7 to 1500 l.
Inuences arising in extraction of EGIcore-HFBI in
detergent-based systems due to the scale of the fungal
cultivations were studied and are also discussed.
2. Materials and methods
The following characteristics describe the partitioning of
molecules in ATPS.
The partition coefcient K is dened as the ratio of con-
centration in the top (c
i,T
) and bottom phase (c
i,B
)
K =
c
i,T
c
i,B
.
The yield in the top phase Y
T
is dened as
Y
T
=
n
T
n
T
+ n
B
or consequently as
Y
T
=
1
1 + ((V
B
/V
T
)(1/K))
If no index is used for the yield it refers to the yield in
the detergent-rich phase.
The volumes of the phases can be described by the volume
ratio R
R =
V
T
V
B
and by the concentration factor in the light phase c
f
c
f
=
V
total
V
T
=
V
T
+ V
B
V
T
where V is the volume of the phase denoted.
2.1. Separation and preparation of ATPS
2.1.1. Separation of phase systems on a laboratory scale
The detergent-based systems in tube scale were produced
by weight using undiluted detergent. The detergent was lled
rst into graduated centrifuge tubes (Assistent Hecht, Merck
KgaA, Darmstadt, Germany). The tubes were then lled
to 10 g with culture supernatant. If salt was added, it was
rst solubilised in the culture supernatant before addition of
the detergent. The tubes were closed and mixed thoroughly
in an overhead shaker (Heidolph, Kehlheim, Germany) at
room temperature. After mixing, the tubes were heated to
the separation temperature and either separated by gravity
settling or by centrifugation at 3000 rpm at constant temper-
ature. After phase separation, the phase volumes could be
determined with a reproducibility of approximately 0.1 ml.
2.1.2. Separation of phase systems on a pilot scale
The separation was performed in two steps.
1st step: To 1160 l culture supernatant with a density of
1005 g/cm
3
50 kg of detergent and 20 kg of NH
4
H
2
PO
4
were added resulting in 4.1% concentration of detergent
K. Selber et al. / Process Biochemistry 39 (2004) 889896 891
and a 0.15 mol/l NH
4
H
2
PO
4
solution. Phase separation was
accomplished by gravity settling, 840 kg of bottom phase
were removed in the 2nd step and 410 kg water and 12 kg
NH
4
H
2
PO
4
were added to the remaining original top phase
so that the salt was solubilised in 300 l of water at 30

C
and added to the bioreactor as well as the remaining 110 l
of water.
Mixing of the phase system during the rst and second ex-
traction was accomplished by stirring gently for 30 min at 60
rpm. In small-scale experiments, the systems have not shown
any tendency to form stable emulsions and they also showed
a very fast mass transport as already described by Fauquex
et al. [17]. Therefore, no problem on a large-scale was ex-
pected. Nevertheless, mixing time and speed were chosen
very conservatively to ensure proper mass transport while
avoiding the creation of microemulsions, which would not
allow a separation by gravity settling. The experiment was
carried out in a bioreactor equipped with a 330 mm Rushton
turbine (New Brunswick Scientic Company, Edison, NJ)
and temperature control set to 24.7 and 30

C, respectively.
2.2. Target protein
The EGIcore-HFBI fusion protein contained the catalytic
domain and the linker region of EGI fused to the gene en-
coding the 75 residue long mature HFBI, thus the cellulose
binding domain of EGI was replaced by HFBI.
2.3. Glycosidase treatment
To 200 l of culture supernatant 200 l of sodium citrate
buffer (buffer 65), 200 l of buffer NP-40, 10 l PNGase F
704S and 10 l Endo H
f
703S (both hydrolases purchased
from Biolabs, Inc., New England, USA) were added. The
samples were incubated at 36

C for 16 h. A reference sam-

ple was treated likewise but omitting enzyme addition. The
separation was carried out using 4% detergent at 30

C.
2.4. Determination of enzymic activity
Endoglucanase I (EGI) was assayed using p-nitrophenyl-
-d-cellobioside (Sigma N-5759) as substrate, dissolved
in 50 mM sodium acetate buffer, pH 5.0. EGI hydrolyses
the -glycosidic bond and p-nitrophenol is released, which
was measured at 400 nm. Since, CBHI (cellobiohydrolase
I) also hydrolyses the substrate it was inhibited using 4.6
mM cellobiose. The reaction is performed at 50

C and
terminated by addition of 12.5% sodium carbonate solution
after 10 min.
4-Methylumbelliferone (Sigma M-1508) was used to gen-
erate a standard curve.
2.5. Cultivations
Production of the target protein EGIcore-HFBI was car-
ried out with a recombinant T. reesei strain VTT D-99702
under the cbhI promoter. The following cultivation media
was used.
20.0 g/l lactose, 4.0 g/l peptone, 1.0 g/l yeast extract,
15.0 g/l KH
2
PO
4
, 2.8 g/l (NH
4
)
2
SO
4
, 0.6 g/l MgSO
4
7H
2
0,
0.8 g/l CaCl
4
2H
2
0, 2.0 ml/l trace element solution [18]
(5.0 mg/l FeSO
4
7H
2
O, 1.6 mg/l MnSO
4
H
2
O, 1.4 mg/l
ZnSO
4
7H
2
O and 3.7 mg/l CoCl
2
7H
2
O).
In the pilot-scale cultivation the amount of lactose was
increased to 40 g/l, all other ingredients remained the
same.
One 200 ml shake ask was inoculated from agar slants
and incubated on a rotary shaker at 200 rpm at 29

C for 2
days. The culture broth was then distributed between three
shake asks and incubated for one more day under the same
conditions. After inoculation of a 15 l bioreactor the culti-
vation was continued for about 4 days at a pH between 4.0
and 5.0 at an agitation rate of 400600 rpm. The pH was au-
tomatically adjusted with sodium hydroxide and phosphoric
acid.
For other bioreactors, the volume of the inoculum was
adapted accordingly. On a 1200 l scale, the temperature at
the beginning was set to 27

C, agitation at 160 rpm and

aeration rate at 12 m
3
/h. The cultivation inoculum was rst
in a 15 l cultivation, then in 180 l each for 24 h. Growth
time in the pilot-scale reactor was 4 days.
Antifoam was injected automatically on foaming.
2.6. Chemicals
All compounds used were of analytical quality.
Agrimul NRE 1205 (C
1218
E
5
) was a gift from Henkel/
Cognis, Dsseldorf, Germany.
The antifoam DOW Corning 1500 (EU), Edegem, Bel-
gium was employed.
Celite 535 (diatomaceous earth) from Celite France SA,
Rueil, Malmeson, France was used as lter aid for rotating
vacuum drum ltration. 12 kg of Celite were suspended
in 250 l water and deposited equally on the drum prior to
ltration.
The vacuum drum lter was constructed by Larox OY,
Lappeenranta, Finland and has a surface area of 1 m
2
.
3. Results
3.1. Scale up of the downstream process
Before the start of the scale up, cultivation was investi-
gated in shake asks and 7 l bioreactors. Downstream pro-
cessing was carried out in 10 ml tube scale. On this small
scale, the basic process was optimised as described in Sel-
ber et al. [19]. All experiments discussed were carried out
using culture supernatant. The inuence of cells is reported
by Selber et al. [19].
The scale up of cultivation and downstream processing
was done using culture broth obtained in large cultivation for
892 K. Selber et al. / Process Biochemistry 39 (2004) 889896
Fig. 1. Comparison of extraction experiments in 1200 l and 10 ml scale.
Phase system composition: detergent (Agrimul NRE 1205) (w/w) =0.041
and 0.15 mol/l (NH
4
)H
2
PO
4
. The extraction temperature for the phase
system was 24.7

C and the pH was 5.5.

the pilot-scale downstreamprocess. The quality of the down-
stream process was judged by a parallel experiment on a test
tube scale, starting with the identical culture supernatant.
At the end of the cultivation, the mycelium was separated
by means of a rotating vacuum drum lter using Celite 535
diatomaceous earth as lter aid. 1160 l was obtained out of
1500 l whole broth.
The supernatant was transferred into the cleaned biore-
actor, adjusted to the separation temperature of 24.7

C and
mixed with salt and detergent to give 0.15 mol/l ammonium
dihydrogen phosphate and 4.1% of detergent Agrimul NRE
1205. The phases were left to separate by gravity settling
for 15 h. The target protein should stay in the light, the
detergent-rich phase. The light phase had a volume of 250
l or a concentration factor of 3.3. The heavier phase was
removed through the bottom valve.
The yield, partition coefcient and concentration factor
from this extraction are shown in Fig. 1. The partition co-
efcient and the concentration factor are equal in the 10 ml
and 1200 l scale separation within the measurement error.
The yield is slightly different due to the small deviations
of partition coefcient and volume ratio. Scale up factor is
1.2 10
5
.
Fig. 2. Separation behaviour of EGIcore-HFBI obtained from 7 l cultivation and pilot-scale (1500 l) cultivation. Extraction was performed with 10
ml culture supernatant each. Phase system composition: detergent (Agrimul NRE 1205) concentration (w/w) =0.041 and 0.15 mol/l (NH
4
)H
2
PO
4
. The
extraction temperature for the phase system was 24.7

C and the pH was pH 5.5.

3.2. Scale up of the cultivation and downstream
process in a two-step procedure
A second-phase separation can be performed to increase
the purity of the fusion protein. Water (additionally salt) can
be added to the detergent-rich phase from the rst separation
step.
In this investigation, a re-extraction was of special in-
terest since endogeneous EGI is expressed in low amounts
by the recombinant host. The activity assay cannot distin-
guish between fusion protein and endogeneous EGI. Endo-
geneous EGI remains almost quantitatively in the bottom
phase.
The cultivation conditions and, therefore, the scale up of
the cultivation have an impact on the overall result as could
be anticipated from the moderate partition coefcients of the
fusion protein and yields presented so far.
The cultivation had to be scaled up from 7 to 1500 l (the
difference to the above mentioned volume of 1200 l is due
to the separation of the mycelia). Even if no major difcul-
ties were expected and encountered during the 11-day cul-
tivation period, in this investigation the inuence of cultiva-
tion can only be judged by the following phase separation.
The results for the extraction from 7 and 1500 l cultiva-
tion are compared in Fig. 2. In a two-step-procedure, new
water and salt were added to the light phase of 250 l to
give a nal volume of 750 l which was stirred and left for
gravity settling for 5 h. The target protein partitioned to the
detergent-rich light phase. The heavy phase was removed
through the bottom valve. As the rst step of the pilot sep-
aration, this second step did not reach the same yield and
partition coefcient as found after cultivation on a 7 l scale.
The apparent K value of 11 and 33 (rst and second sep-
aration, respectively, 7 l) and 3.8 and 7.7 (rst and second
separation, respectively, 1500 l) demonstrate the signicant
differences.
Several factors which could cause experimentally ob-
served differences were investigated: insufcient mass
transport, high entrainment, inuence of antifoam, genetic
K. Selber et al. / Process Biochemistry 39 (2004) 889896 893
instability of the strain, differences in the cultivation proce-
dure and glycosylation of the fusion protein.
3.3. Limiting mass transport/insufcient mixing
While stirring the culture supernatant slowly, salt was
solubilised in the bioreactor and detergent was added. The
bioreactor used as mixer and settler was stirred at 60 rpm
for 30 min while the temperature was maintained at 24.7

C.
The long mixing time should ensure a sufcient mass trans-
fer while a slow rotational speed was chosen to prevent
the formation of stable microemulsions which would lead
to a very slow separation. Parallel tube scale investigations
showed that mass transport was satisfactory.
3.4. Entrainment/insufcient separation
High entrainment, especially entrainment in the bottom
phase lowers the apparent partition coefcient and yield.
Entrainment was monitored in the rst and second separa-
tion step. In the rst step, the entrainment (incomplete sep-
aration) could be observed mainly in the top phase, which
included 16% of bottom phase. In the second step, no en-
trainment could be monitored in the top phase while 4.6%
of top phase remained in the bottom phase. Assuming the
entrained phases were entrapped after the transport of fu-
sion protein was completed the amount of fusion protein in
the entrainment can be considered as high as its amount in
the puried phases. The theoretical separation results in the
absence of entrainment can be calculated. The results are
shown in Table 1.
The increase in concentration factor from 3.4 to 3.8 and in
K of 3.8 to 4.2 and no difference in yield cannot explain the
big difference between the separation experiments shown in
Fig. 2. The yields in the presence of entrainment and in the
absence of entrainment are almost identical since mainly
heavy phase was entrapped in the light phase.
It has to be mentioned that, while performing the mea-
surement of the entrainment volume, no entrainment could
be measured after 10 min of centrifugation in 10 ml cen-
trifuge tubes at 3000 rpm. It appears that the emulsion is
stabilised by ingredients from the culture broth. This inter-
pretation is enforced by samples taken from the very top
(and from the very bottom) of the phases in the large-scale
experiment which show no higher (lower) activity than the
phase average (data not shown).
Table 1
The calculated effect of the entrainment on the concentration factor,
partition coefcient and yield of EGI activity in the 1200 l separation
experiment
Concentration
factor, c
f
Partition
coefcient, K
Yield,
Y (%)
Measured values
with entrainment
3.4 3.8 59
Calculated values
without entrainment
3.8 4.0 58
3.5. Inuence of antifoam
Antifoam was automatically injected into the bioreactor
during cultivation. No device for precise measurement of
the injected antifoam volume was available. Therefore, the
amount of antifoam could only be estimated.
In the pilot-scale cultivation a different antifoam than in
the 7 l cultivation was used, however, no inuence on the sep-
aration data was expected from shake ask controls where
partition experiments were performed with separately added
antifoam in different concentrations (not shown).
3.6. Genetic instability
This cultivation was the rst in which the producing strain
was grown in such a large volume. The total growth period
from the original agar slant was an uninterrupted 7 days.
At the end of the cultivation, the strain was routinely
plated on potato dextrose agar to monitor the growth and
especially the sporulation of the fungus. Two different phe-
notypes of the spores could be observed on the plates. Plates
were prepared using different dilutions of the mycelium.
They show a usual dark green and an unusual light green
colour. In other cultivations only one phenotype occurred.
To investigate a possible inuence the spores were iso-
lated, germinated, grown in shake asks and the culture
broth extracted. The resulting partition coefcients for
EGIcore-HFBI under the same extraction conditions as in
the pilot-scale experiment were K=37 and 26, respectively.
3.7. Protein instability
The hydrophobin directs the partitioning of the fusion
protein [20,15] Hydrophobin could potentially be cleaved
fromthe active endoglucanase core thus altering the partition
results since endoglucanase activity is measured. However,
the fusion protein was not degraded in the culture broth, as
controlled by SDS-PAGE and Western blot.
3.8. Extraction of samples during cultivation
Samples were taken routinely during the cultivation pro-
cess after inoculation of the 1500 l bioreactor. These sam-
ples were extracted at 30

C with 4% detergent. Due to the

higher temperature the values were different to the values
in the pilot-scale experiment ([20,15]). At 13 h of growth
a partition coefcient of 10 was observed. After 25 h of
cultivation, the partition coefcient dropped signicantly to
values lower than 4 and decreased to 2.5 after 94 h. A sig-
nicant lag phase was not detected as seen, e.g. by protein
concentration measurements during cultivation (not shown).
3.9. Glycosylation
To investigate the glycosylation of the fusion protein
and its inuence on the partitioning, culture supernatant
894 K. Selber et al. / Process Biochemistry 39 (2004) 889896
Table 2
Extraction experiments using culture supernatant produced in different
scale and at different temperatures
Cultivation
temperature (

C)
c
f
K Y (%)
7 l cultivation 29 3.3 11 81
1st pilot scale 2227 3.4 3.8 59
7 l cultivation 2227 3.3 7.9 78
2nd pilot scale 29 2.8 8.3 81
EGI activity is measured.
from the pilot-scale fermentation was treated with the gly-
cosidases PNGase F and Endo H
f
. Because of the high
protein content in the supernatant and the small amount of
glycosidases available only partial deglycosylation could
be expected. Separating the treated supernatant using 4.0%
detergent without adding salt resulted in a more than dou-
ble partition coefcient compared to the untreated sample
(K(deglycosylated) =5.9; K(not treated) =2.5).
SDS-PAGEs of large and small scale cultivations do not
show large difference in glycosylation, however, there was
a large distribution in glycosylation (data not shown).
An additional small inuence on the partition coefcient
by the deglycosylation of endogenous EGI is possible. How-
ever, the amount of endogeneous EGI is relatively small and
was calculated to 1/7 of total EGI [20].
3.10. Scale and temperature of the cultivation
Cultivation on a large-scale was conducted slightly dif-
ferent from the cultivations which served to optimise the
separation procedure, leading to differences in pH, dissolved
oxygen and growth characteristics. Thus, the cultivation
on a larger scale was conducted at a starting temperature
of 27

C and reduced in two steps to increase protein pro-

ductivity. To exclude the inuence of these proles a very
similar prole was run in a 7 l cultivation. The tube scale
extraction results of this cultivation are shown in Table 2.
The measured partition coefcient of 7.9 is about twice as
high as in the pilot-scale experiment. A second pilot-scale
cultivation was carried out in the same experimental set up,
but this time 29

C was chosen as cultivation temperature.

With regard to temperature the cultivation was similar to
the cultivations carried out during optimisation [19]. The
resulting partition coefcient of 8.3 is again smaller than in
the optimised case (K=11), but signicantly higher than
the rst pilot-scale cultivation (K=3.5).
4. Discussion
4.1. Scale up
Kroner et al. [4,21] showed that scale up of extraction
processes in ATPS is easy and well predictable for PEG/salt
systems. This was proven by gravity settling experiments
as well as by centrifugal separation using disk stack sepa-
rators. Patents report successful operation in large-scale in
industry [3].
The largest detergent-based gravity settling experiment
found in the literature was conducted on a 5 kg scale. No
comparison to the tube scale experiment is reported [11].
This investigation demonstrates the ease of up scale of
detergent-based systems with respect to gravity settling.
The unusual high scale up factor of 1.2 10
5
for a single
step emphasises the cheap, reliable and unproblematic scale
up of the downstream process. Centrifugation has been
demonstrated not to be the optimal means for separation of
detergent-based systems due to the low interfacial tension
and the high friction in the separator, which hinders the
essential coalescence of droplets [12].
4.2. Limited mass transport
Mass transfer in detergent-based systems is very fast
resulting from a very low interfacial tension and, there-
fore, generation of small droplets and large surface area
at low-energy input [17] According to Minuth et al. [11]
the interfacial tension is about 0.010.004 mN/m. This is
a much smaller value than, for example, the interfacial
tension of PEG/phosphate systems (0.040.1 mN/m [17]).
A series of separation experiments were conducted on a
tube scale to ensure complete mass transfer. Since, paral-
lel experiments on a 10 ml scale led to the same result as
the large-scale experiment incomplete mass transfer can be
ruled out as a reason for the low values found.
4.3. Entrainment/insufcient separation
The entrainment observed is undesired, however, it does
not contribute signicantly to lowering of the experimen-
tal values and can easily be overcome. The reason for the
entrainment in the bottom phase in the second extraction
step is the fact that the required settling time could not be
monitored in the bioreactor, because an observation window
was lacking. In a large-scale process this could be over-
come by separation of the total volume or re-separation of
the smaller volume close to the interface in an appropriate
vessel geometry including an observation window or using
a transparent vessel.
If desired, the separation time can be shortened signi-
cantly by the use of static mechanical devises like coalesces.
Due to friction problems leading to redispersion dynamic
devices have to be treated with caution as indicated above.
It has to be mentioned that detergent-based ATPS can
cause problems in phase separation if microemulsions are
formed. This was demonstrated by Minuth et al. [11] and
Selber [20] on a smaller scale. The reason for formation of
microemulsion was ascribed to the presence of additional de-
tergent or amphiphilic compounds under cultivation condi-
tions. Hydrophobins like HFBI and HFBII, expressed by the
recombinant host, may have inuenced the large-scale exper-
iment, but no phase separation problems were encountered.
K. Selber et al. / Process Biochemistry 39 (2004) 889896 895
4.4. Inuence of antifoam
Antifoams are amphiphilic detergents themselves. They
can interact with the micelles of the phase building de-
tergent and they can interact with the amphiphilic protein
EGIcore-HFBI. Little is known about the interactions with
proteins, but the inuence on the binodal has been described.
By addition, especially of amphiphilic substances the phase
diagram is altered [22,23] and the partitioning of proteins
can become less efcient.
Here, the amount of antifoam employed was in the range
0.2% of total volume. Even if only small additions of sub-
stances can have a signicant inuence, the amount of an-
tifoam was too small to cause the differences in extraction.
4.5. Genetic instabillity
The partitioning values of re-grown T. reesei cultures were
much higher than in the initial pilot-scale experiment, cor-
responding to previous shake ask cultivations. However,
there was a signicant difference in colour between the two
phenotypes found. This could be due to different sporulation
environment, but it could also be a result of the screening
procedure since the strain is not monoclonal. Due to the dif-
cult biochemical and genetic work necessary to understand
the molecular differences in the strain the investigation of
this topic was not pursued further.
Besides for partition coefcients >20 small analytical de-
viations determining the concentration/activity in the heavy
phase will lead to a larger error, and the difference found in
K values of 37 and 26 may not be signicant.
4.6. Glycosylation
Glycosylation in general can have a signicant inuence
on the partitioning of proteins as described by Pryde and
Philipps [24] for membrane proteins or by Terstappen et al.
[25] for lipases. The fusion protein EGIcore-HFBI includes
the linker region connecting the catalytic core with HFBI
replacing the cellulose-binding domain. The linker of wild
type EGI of T. reesei is highly glycosylated [26], while the
hydrophobins are not glycosylated. The glycosylation of the
linker is known to have a broad distribution, as glycosylation
in general.
Obviously, the glycosylation reduces the driving force of
partitioning to the detergent-rich phase. This can be true for
different reasons: the sugar chains extend into the solvent
and hinder the enclosure of the protein by a detergent mi-
celle. Glycosylation can hinder the approach of micelles to
a protein or affect the agglomeration behaviour of proteins.
This can additionally lead to a change of the optimal param-
eter for separation.
The other possibility is that glycosylation might hide the
hydrophobic parts of the enzyme which play an important
role in the driving force for separation [25]. A change in
activity of highly glycosylated enzymes is not expected as
the glycans do not inuence enzymesubstrate interaction
[27].
The glycosylation distribution can in principal be inu-
enced and narrowed by the cultivation procedure. Therefore,
the negative inuence of excess glycosylation on the parti-
tioning can be overcome by an well-adapted and controlled
cultivation process. Another possibility would be to delete
the most glycosylated region of EG1-the linker-completely
or partially. Theoretically, a procaryotic host could be used
for production to cancel out any glycosylation. Unfortu-
nately, bacterial systems do not match Trichoderma in their
secretion capacity.
The change in partitioning can also not be related to a
change in growth rate. The growth rate has an inuence e.g.
on the glycosylation. The growth in pilot-scale was very
steady and no dened lag phase could be observed.
A long time of unlimited growth, starting from the
pre-cultures and taking up to 11 days until the end of the
pilot-scale cultivation, could cause the difference in parti-
tioning, due to a change of the characteristics of the protein,
e.g. by a change in glycosylation.
4.7. Scale and temperature of the cultivation
From these experiments it is obvious that the size and
temperature of the cultivation must have a signicant inu-
ence, even if it might not be the only one.
The temperature has a strong inuence as could be demon-
strated in the second pilot-scale experiment which showed
signicantly higher values for the partition coefcient and
the yield in comparison to the rst pilot-scale experiment at
lower temperature.
Since, the partition coefcient of the rst 7 l cultivation
at 25

C was higher than in the corresponding and second

pilot-scale cultivation (the yields were similar due to the dif-
ferent concentration factor) the temperature cannot be the
only effect. This is emphasised by the equivalent investiga-
tion at low temperatures.
The second reason must be linked to the scale of the
cultivation. If the size of the cultivation had a strong effect,
it would indicate that most likely either the total growth
time or the transport mechanism of metabolic substances,
e.g. the oxygen transfer towards the cells causes the change
in separation behaviour of the fusion protein. This can
additionally be emphasized through very high values of
partition coefcients and yields in experiments form shake
ask cultivations.
5. Conclusion
The technical feasibility of large-scale extractions in
detergent-based ATPS was proven, however, care must be
taken in the scale up of the cultivation process that prop-
erties of the target protein are not altered. This appears
especially important for glycosylated protein.
896 K. Selber et al. / Process Biochemistry 39 (2004) 889896
Acknowledgements
We would like to thank our colleagues from VTT, Finland
who supported the experimental work. We also like to thank
Hans Christian Raths, Horst Wollenweber and Jean-Pierre
Moiltor Henkel/Cognis for the generous supply of Agrimul
NRE 1205. The work has been supported by a grant from
the EU-Project BI04-CT96-0435.
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