Purification of Recombinant Protein A by
Aqueous Two-Phase Extraction Integrated
with Affinity Precipitation
Masamichi Kamihira,'*** Rajni Kaul,' and Bo Mattiasson'
'Department of Biotechnology, Chemical Center, University of Lund, Lund,
Sweden; and 2Department of Chemical Engineering, Faculty of Engineering,
Nag0ya University, Nag0ya, Japan
Received February 4, 1992/Accepted July 24 1992
Aqueous two-phase extraction incorporated affinity pre-
cipitation was examined as a technique for protein purifica-
tion. A n enteric coating polymer, Eudragit SIOO, was
employed as a ligand carrier. Eudragit was specifically par-
titioned t o the top phase in the aqueous two-phase sys-
tems. For application of this method to purification of
recombinant protein A using human IgG coupled to Eu-
dragit in an aqueous two-phase system, 80% of protein A
added was recovered with 81% purity. The purity was en-
hanced 26-fold by this method. The IgG-Eudragit could be
used repeatedly for the purification process. This separa-
tion method should be applicable to industrial-scale purifi-
cation as a new purification procedure combining the
advantages and compensating for the disadvantages of the
aqueous two-phase method and affinity precipitation
method. 0 1992 John Wiley 81Sons, Inc.
Key words: aqueous two-phase extraction affinity precipi-
tation Eudragit protein A
INTRODUCTION
Advances in genetic engineering and cell fusion tech-
niques have made possible the realization of commercial-
scale production of various physiologically active
substances such as hormones and vaccines. However,
the separation techniques for such bioproducts have
been slow in development compared with the produc-
tion techniques. This is one of the latest problems in
biotechnology.16 Conventionally, bioproducts have been
purified by precipitation methods using salts and/or or-
ganic solvents and various column chromatography pro-
cedures. The chromatographic operations are sometimes
difficult for industrial-scale purification and the need for
the development of new, effective separation techniques
has been felt.
Liquid-liquid extraction in the aqueous two-phase
system has provided a gentle and scalable procedure for
the separation of various biological substances.' In this
method, if the substance of interest is extracted to the
top phase, it can be separated with minimal energy re-
quirement from cells and cell debris, which usually par-
tition to the bottom phase. Commonly used aqueous
two-phase extraction by spontaneous partitioning may
* To whom all correspondence should be addressed at the De-
partment of Chemical Engineering, Faculty of Engineering,
Nagoya University, Chikusa-ku, Nagoya, 464-01, Japan.
Biotechnology and Bioengineering, Vol. 40, Pp. 1381-1387 (1992)
0 1992 John Wiley & Sons, Inc.
not extract a protein specifically from a crude mixture.
To overcome this problem, the introduction of an affin-
ity ligand into one of the phases (usually polyethylene-
glycol) has been attempted to enhance the s p e c i f i ~ i t y . ~ ~ ~ ~ ' ~
However, this procedure still has some limitations in
the effective removal of impurities and recovery and
reuse of the ligand.
During the past decade, another potentially scalable
technique, that of affinity precipitation, has been studied
for the isolation of protein^.'^*'^,'^ The affinity ligand,
coupled to a reversibly soluble-insoluble polymer, is
used to specifically bind the protein in a complex mix-
ture. The precipitation step, which can be accomplished
by a change in any environmental parameter such as pH,
temperature, salinity, etc., facilitates the separation of
the bound ligand and affinity complex from the unbound
components. Although cells and cell debris should be re-
moved before use, recovery and reuse of the ligand are
relatively easy, and highly specific and scalable separa-
tion can be expected by this method.
In the present study, we describe a new separation
procedure using aqueous two-phase separation method
incorporated affinity precipitation, in order to combine
the advantages and compensate for the problems in both
the methods described above. This method was applied
for the purification of protein A produced by recombi-
nant Escherichia coli as a model system.
MATERIALS AND METHODS
Materials
An enteric coating polymer, Eudragit (S 100 type, co-
polymer of methacrylic acid and methylmethacrylate; a
commercial product of Rohm Pharma GmbH, Darm-
stadt, Germany), was used as a reversibly soluble-
insoluble ligand carrier. The polymer has been used for
coating of drugs, since the solubility in water can be
controlled by pH change; i.e., while it is insoluble in
gastric juice, it dissolves in the region of the digestive
tract where the juices are neutral to weakly alkaline. Re-
cently, Eudragit was used as a polymer matrix for the
immobilization of enzymes acting on macromolecular
CCC 0006-3592/92/01101381-07
substrates." The enzymatic reaction could be per-
formed with the immobilized enzyme in a soluble form,
which could later be recovered by precipitation. The
present study was undertaken to study the possibility of
using Eudragit as a ligand carrier for affinity partition-
ing of proteins in aqueous two-phase systems, in a man-
ner that the bound ligand can be easily recovered by
precipitation and the elution of the protein from the af-
finity complex can be independently controlled.
Polyethylene glycol (PEG) 8000 (Union Carbide Co. ,
New York, NY) and hydroxypropyl starch, Reppal PES
ZOO9 (Reppe Glykos AB, Vaxjo, Sweden), were used as
polymers for making aqueous two-phase systems. Pro-
tein A isolated from the Staphylococcus aureus cell wall
was obtained from Sigma Chemical Co. (St. Louis,
MO), and human IgG was purchased from KabiVitrum
AB (Stockholm, Sweden).
Immobilization of Human IgG to Eudragit
Human IgG was coupled to Eudragit using water-soluble
carbodiimide. The immobilization procedure was as
follows: 0.25 g of Eudragit was suspended in 25 mL of
0.01M phosphate buffer solution containing 0.14M NaCl
(PBS), and the pH of the solution was adjusted to 7.2 by
adding 2N NaOH to solubilize the polymer. To this
solution 0.075 g of 1-et hyl-3-(3-dimethylaminopropyl) -
carbodiimide hydrochloride (Sigma Chemical Co.) was
added under stirring. After 10 min, 0.5 to 1 mL of hu-
man IgG solution (15 to 120 mg/mL), previously dia-
lyzed against PBS, was added.
The coupling mixture was allowed to stand for 3 h at
room temperature with constant stirring. Then, 0.7 mL
of 0.45 g/mL ethanolamine solution (pH 8.5) was added
for blocking of the remaining activated sites on the poly-
mer. After incubation for 1 h, the pH of the mixture
was adjusted to 4.5 by adding 2M acetic acid solution
and the precipitated IgG-Eudragit complex (IgG-
Eudragit) was collected by centrifugation and washed
three times with 25 m L of 0.01M acetate buffer
(pH 4.5). The IgG-Eudragit precipitate was dissolved in
potassium phosphate buffer and the pH of the solution
was adjusted to 7.0. The final solution contained 1%
Eudragit, 0.2M potassium phosphate and 0.05% sodium
azide for preservation, and was stored at 4°C.
pH Response Test of Eudragit
Because all Eudragit was precipitated at pH less than
3.5 and the amount of the precipitate was almost pro-
portional to the turbidity, absorbance at 470 nm was
measured to study the precipitation response of Eu-
dragit to variations in pH, as reported by Taniguchi
et al.17,'8Various amounts (3 to 150 pL) of 2M acetic
acid solution was added to 3 mL of 0.15% Eudragit or
IgG-Eudragit solution, and the mixture was well-mixed.
After stabilization of the turbidity, the pH and the ab-
1382 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 11, DECEMBER 20, 1992
sorbance at 470 nm for each solution were measured. A
ratio of the absorbance at each pH to the maximum
value of the absorbance at pH less than 3.5 was calcu-
lated as relative value.
Evaluation of Adsorption Capacity
Adsorption capacity with respect to the amount of pure
protein A adsorbed to IgG-Eudragit was determined as
follows: 0.5 mL of 0.5% IgG-Eudragit solution and
0.5 mL of 20% PEG 8000 solution were mixed, and
100 pg of pure protein A was added to the mixture.
After overnight incubation at 4"C, the pH of the mix-
ture was adjusted to 4.5 by adding 2M acetic acid. After
centrifugation, the supernatant was collected and dia-
lyzed against PBS to remove PEG. The amount of
protein A in the dialyzed solution was measured. Ad-
sorption of protein A to Eudragit treated only with
ethanolamine was measured by the same procedure.
This amount was regarded as nonspecific adsorption to
Eudragit. Adsorption capacity of IgG-Eudragit was cal-
culated by subtracting the amount of nonspecific ad-
sorption from the amount adsorbed to IgG-Eudragit.
Preparation of Recombinant Protein A
Crude Extract
E. coli N4830-1 (Pharmacia Fine Chemical Co., Uppsala,
Sweden), harboring a plasmid pRIT2T (Pharmacia)
encoding S. aureus protein A was cultivated in a me-
dium (pH 7.0) containing, per liter: 10 g bacto-tryptone,
5 g yeast extract, 5 g NaCI, 1 g glucose, and 80 mg ampi-
cillin. The protein A gene was located to the down-
stream of PRpromoter derived from A-phage. Therefore,
production of the protein A was heat-inducible, and the
product was accumulated in the cells. The cells were
grown first at 37°C until the absorbance at 600 nm
reached approximately 2.5. Thereafter, the temperature
was raised to 42°C and the cultivation was continued
further for 4 h. The protein A content in total soluble
protein of the cell was 3.1%. The cells were collected by
centrifugation and stored at -20°C until use. Protein A
crude extract was prepared by sonication of the E. coli
cell suspension in 0.2M potassium phosphate buffer
(pH 7.0). The lysate was directly used for purification
without elimination of cell debris.
Purification Procedure
PEG 8000, Reppal PES 200, 1%IgG-Eudragit solution,
0.4M potassium phosphate solution, and the E. coli cell
homogenate were mixed to form an aqueous two-phase
system having a final concentration of 5% PEG 8000,
14% Reppal PES 200, 0.5% IgG-Eudragit and 0.2M
potassium phosphate. After occasional stirring for 1 h,
the mixture was centrifuged at 4000 rpm for 10 min,
and the top phase and the bottom phase were sepa-
rated. The top phase was used to recover the protein A. Rabbit muscle phosphorylase b (MW 97,000), bovine
The pH of the top-phase solution was adjusted to 4.5 by serum albumin (MW 66,000),ovalbumin (MW 45,000),
adding 2M acetic acid, and the resulting precipitate was bovine carbonic anhydrase (MW 31,000), soybean tryp-
collected by centrifugation at 4500 rpm for 10 min. sin inhibitor (MW 22,000), and hen egg white lysozyme
The precipitate was then washed three times with (MW 14,400), available as a standard kit (Bio-Rad Labo-
0.01M acetate buffer (pH 4 3 , and later resuspended in ratories Ltd., Richmond, CA), were used as molecular
2 mL of 0.1M glycine-HCI buffer (pH 2.5) to elute pro- weight markers.
tein A from IgG-Eudragit. The elution procedure was
repeated twice. Each fraction of eluate was neutralized
RESULTS AND DISCUSSION
immediately with 0.5 N NaOH. The precipitate (in-
soluble form of IgG-Eudragit) was washed with 0.01M
Partition Behavior of Eudragit in the Aqueous
acetate buffer (pH 4.5) and resolubilized in 0.2M potas- Two-Phase System
sium phosphate buffer (pH 7.0). This solution was used
for the next cycle of the purification. The partition behavior of Eudragit in the PEG 8000-
Reppal PES 200 two-phase system was examined. In
this study, modified starch, Reppal PES 200 was used as
Analyses hydrophilic bottom-phase polymer instead of dextran,
Eudragit concentration was estimated by measuring the because the material is rather inexpensive and promis-
turbidity at 470 nm after precipitation of the polymer ing for industrial-scale use.
and calculating the amount using calibration curves. For Table I shows Eudragit concentrations in the two-
the measurement of recovery of Eudragit, the precipi- phases as a result of change in the initial concentration
tate collected after centrifugation was washed three of the polymer. Most of the Eudragit was partitioned to
times with 0.05M acetic acid, dried overnight at llO"C, the top phase in every case, although Eudragit concen-
and then weighed. tration of the bottom phase was increased with the in-
Phase diagrams (binodial curves) were determined by crease in the initial concentration. Moreover, Eudragit
the turbidity method described by Albertsson.' For the from the top phase was recovered almost completely af-
two-phase systems containing Eudragit, predetermined ter precipitation. Also, in the other aqueous two-phase
concentrations of Eudragit were added to each solution systems such as PEG-dextran and PEG-phosphate, most
for making the binodials. of the Eudragit was partitioned to the top phase (data
Total protein concentration was determined by the bi- not shown). It is, therefore, possible that Eudragit could
cinchoninic acid method described by Smith et a1.I5 Bo- be used as a ligand carrier to partition the protein of
vine serum albumin or protein A was used as a standard. interest specifically to the top phase in the aqueous two-
Protein A concentration was determined by a solid- phase systems.
phase enzyme-linked immunosorbent assay method The effect of Eudragit addition on the phase diagram
(ELISA).3The wells of the microtiter plate (Coster Co., is shown in Figure 1, and the lines represent phase-
Cambridge, MA) were coated overnight at 4°C with separation limit. The phase separation was promoted by
mouse anti-protein A monoclonal antibody (Sigma the addition of Eudragit to the two-phase system. How-
Chemical Co.). The plate was washed three times with ever, the shape of the binodial was not changed much,
PBS containing 0.05% Tween 20. After addition of the even beyond Eudragit concentration of 0.25%.
protein A samples to the wells, the plate was allowed to
stand for 30 min. Table I. Partition and recovery of Eudragit S 100 in PEG 8000-
After washing three times with PBS containing 0.05% Reppal PES 200 two-phase system.
Tween 20, the diluted solution of rabbit IgG conjugated Concentrationa (wt%) Recovery from
to horseradish peroxidase (Dakopatts A/S, Glostrup, Initial concentration top phaseb
Denmark) was added to the wells and the plate was (wt%) TOP Bottom (%)
stood for 1 h. Then the wells were washed four times 0.1 0.20 ND' 101
with PBS containing 0.05% Tween 20 and added the (100%) ( 0 %)
substrate solution containing H 2 0 2and 2,2'-azino-bis 0.25 0.59 0.0007 104
(3-et hylbenz-thiazoline-6-sulfonicacid) diammonium to (99.9%) (0.1%)
detect the enzyme. The protein A concentration was es- 0.5 0.86 0.025 103
(97.7%) (2.3%)
timated by measuring absorbance at 405 nm using an 1.0 1.38 0.31 103
ELISA reader (Labsystems OY, Helsinki, Finland). The (85.0%) (15.0%)
commercial protein A or the recombinant protein A pu-
rified by IgG-sepharose was used as a standard. The composition of the phase system was 5 wt% and 14 wt% of
PEG 8000 and Reppal PES 200, respectively.
Sodium dodecyl sulfate-polyacrylamide gel electro- a Estimated from turbidity at 470 nm after precipitation.
phoresis (SDS-PAGE) with 13% gel was performed ac- Estimated from dry weight after precipitation.
cording to Laemmli' to check the purity of the samples. Not detected.

KAMIHIRA, KAUL, AND MATTIASSON: AQUEOUS TWO-PHASE WITH AFFINITY PRECIPITATION 1383
 h '"1 1
Table 111. Effect of salt addition on partition of Eudragit S 100.

Eudragit
concentrationa
(wt%)
Concentration
(MI TOP Bottom

Salt + 0.5% Eudragit

No addition 0.86 0.025
(97.7%) (2.3%)
0 5 10 15 20 K phosphateb 0.05 0.84 0.005
Reppal PES 200 (wt%) (99.5%) (0.5%)
Figure 1. Phase diagrams of PEG 8000 and Reppal PES 200 con- 0.2 1.02 ND'
taining Eudragit S 100. Symbols: 0, 0%; 0, 0.1%; A, 0.25%; A, (100%) (0%)
0.5%; 0, 1.0%. NaCl 0.2 0.67 0.24
(76.5%) (23.5%)
The effect of the phase composition on the partition 0.5 0.39 0.56
(43.8%) (56.2%)
of Eudragit is shown in Table 11. Because each composi- Salt + 1.0% Eudragit
tion examined was almost along the same tie line, it No addition 1.38 0.31
could be considered that the compositions of each top (85.0%) (15.0%)
and bottom phase were the same. Consequently, the re- K phosphateb 0.05 1.66 0.059
(97.2%) (2.8%)
sults show the effect of volume ratio between the phases
0.2 2.27 0.041
on the Eudragit partitioning. The partition to the bot- (98.2%) (1.8%)
tom phase was increased with the decrease of the top- NaCl 0.2 1.28 0.77
phase volume. Furthermore, the partition to the bottom (66.6%) (33.4%)
phase was drastically increased as top phase concentra- 0.5 0.59 1.31
(31.7%) (68.3%)
tion of Eudragit exceeded 1% (Tables I and 11).
The influence of salt addition on the partition of Eu- The composition of the phase system was 5 wt% and 14 wt% of
dragit was also studied (Table 111). The experiments PEG 8000 and Reppal PES 200, respectively.
a Estimated from turbidity at 470 nm after precipitation.
were performed with 0.5% and 1% Eudragit concentra-
K phosphate: equal molar mixture of KH2P04 and KzHP04.
tions, in which the partition to the top phase was less Not detected.
than 98%. It is apparent from the data that the partition
to the top phase was increased by the addition of potas- phosphate ion strongly favors the bottom phase and
sium phosphate. When 0.05M phosphate was added to chloride ion is present equally in the both phases,6 elec-
the two-phase system, Eudragit partition to the top trostatic repulsion between Eudragit, polyanion in the
phase was increased to 99.5% for 0.5% Eudragit and soluble form, and the salt anions generates the parti-
97.2% for 1% Eudragit. With the increase in phosphate tioning. Thus, the addition of anion specifically parti-
concentration to 0.2M almost all of the 0.5% Eudragit tioned t o the bottom phase will be effective for
and more than 98% of the 1% Eudragit was partitioned complete recovery of Eudragit from the top phase.
to the top phase. On the other hand, the addition of
NaCl had the opposite effect. More than 50% of Eu-
dragit added was partitioned to the bottom phase for Character of Human IgG-immobilized Eudragit
both concentrations of Eudragit when 0.5M NaCl was The protein A and IgG-binding system was selected as a
added. This seems to be attributable to the distribution model system for the application of this purification
of the anion in the two-phase system. From the fact that procedure. For this, human IgG was covalently coupled
Table 11. Effect of phase composition on partition of Eudragit to carboxyl groups of Eudragit by the carbodiimide
s 100. method in order to be used for the purification of pro-
tein A. For all the concentrations of IgG used, more
Composition Eudragit than 90% of the antibody added was immobilized to Eu-
concentrationa (wt%)
PEG 8000 Reppal PES Volume ratio dragit (data not shown).
(wt%) 200 (wt%) TOP Bottom (top/bottom) Figure 2 shows the precipitation response of Eudragit
and IgG-Eudragit with change in pH. Native Eudragit
2 25 1.81 0.21 0.22
(65.4%) (34.6%) had a soluble-insoluble transitional region between
5 14 0.86 0.025 1.2 pH 4.5 and 5.5. The region was shifted slightly upward
(97.7%) (2.3%) in pH scale after the immobilization of human IgG.
8 6 0.57 0.048 5.3 However, the response width was not changed. Even
(98.4%) (1.6%) though the IgG density immobilized was increased to
Initial concentration of Eudragit was 0.5 wt%. 400 mg of IgG/g of Eudragit, the pH response was al-
aEstimated from turbidity at 470 nm after precipitation. most the same as with the lower densities. The shift in

1384 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 11, DECEMBER 20, 1992

3 4 5 6 7 8
PH
Figure 2. Precipitation curve of Eudragit S 100 and IgG-Eudragit
in response to pH variation. The different samples studied con-
tained: Eudragit only ( 0 ) ;60 mg of IgG/g of Eudragit (A); 240 mg
of IgG/g of Eudragit (+); 400 mg of IgG/g of Eudragit (m); Eudra-
git + 10% PEG (0);120 mg of IgG/g of Eudragit + 10% PEG (A);
and 400 mg of IgG/g of Eudragit + 10% PEG (0).
p H response curves was also obtained when just
ethanolamine or bovine serum albumin were coupled to
Eudragit (data not shown). Thus, the independent pre-
cipitation character on the kind or density of ligand is
important for general use.
The pH response in the presence of PEG was also
examined (Fig. 2), because about 10% of PEG was pre-
sent in the top phase of the two-phase system used in
this study. The pH response behavior for IgG-Eudragit
was the same both in the absence and presence of PEG.
However, the pH response curve for native Eudragit was
shifted upward in the presence of PEG and overlapped
with the curves for IgG-Eudragit.
Next, protein A binding activity of IgG-Eudragit was
examined. Some proteins were adsorbed nonspecifically
to Eudragit (data not shown), and also 80% of protein A
added was adsorbed to Eudragit. This is a potential prob-
lem if Eudragit is to be used in affinity precipitation
method. It is probably partly due to the hydrophobic
character of the polymer. Fortunately, this nonspecific
adsorption was reduced very much in the presence of
PEG, including the nonspecific adsorption of protein A,
which was repressed to more than 90% in 10% PEG
solution. Because, in our studies, the top phase of the
two-phase system contained about 10% PEG, the use
of Eudragit in the aqueous two-phase system had a
great advantage in terms of prevention of nonspecific
adsorption.
Figure 3 shows adsorption of protein A t o IgG-
Eudragit with respect to the change in IgG density. The
amount of protein A adsorbed to IgG-Eudragit increased
with the increase in the amount of immobilized IgG.
However, the increase in the amount was not propor-
tional as obvious from the binding ratio of mol-protein
A adsorbed to mol-IgG immobilized. For instance, the
binding ratio for 30 mg of IgG/g of Eudragit was 0.5, but
the value was decreased to 0.21 for 400 mg of IgG/g of
Eudragit. These results were similar to reported by
Taniguchi et al.," in which they used another pH re-
sponsive support, hydroxypropylmethylcellulose acetate,
KAMIHIRA, KAUL, AND MATTIASSON: AQUEOUS TWO-PHASE WITH AFFINITY PRECIPITATION
- 30 i r 1.0
Immobilized IgG density
(mglg-Eudragit)
Figure 3. Adsorption capacity and binding ratio of IgG-Eudragit
for pure protein A. Symbols: 0, adsorbed protein A; 0, binding
ratio.
for immobilization of human IgG. It seems that high
density of immobilized IgG caused steric hindrance for
protein A adsorption.
Aqueous Two-Phase Partitioning and Affinity
Precipitation of Protein A
Recovery of protein A from an aqueous two-phase sys-
tem was examined. The protein A was partitioned spon-
taneously in the aqueous two-phase system (5% PEG
8000, 14% Reppal PES 200, and 0.2M potassium phos-
phate) with a partition coefficient of 0.85. This was fol-
lowed by the partitioning of the protein A in the system
containing IgG-Eudragit. Based on the results in Fig-
ure 3,100 pg and 200 pg of protein A were added to the
two-phase systems containing 0.025 g of IgG-Eudragit
(60 mg of IgG/g of Eudragit). The results are shown in
Table IV. The apparent partition coefficient calculated
from the amount of protein A remaining in the bottom
phase was found to be 28 and 2.6, respectively. Protein A
amount recovered from the fractions of eluate was al-
most the same in both cases, and the recovery was 91.7%
and 63.0%, respectively. A large amount of protein A
was detected in the PEG phase after precipitation for
the 200 pg addition. From the results, adsorption capac-
ity of the IgG-Eudragit in the two-phase was calculated
as about 4 mg of protein A/g of Eudragit.
Table IV. Recover of purified protein A from PEG 8000-Reppal
PES 200 two-phase system containing IgG-Eudragit.
Added Recovered protein A (pg)
protein A Recovery"
(PgLp) El E2 Bottom (%I
100 75.9 15.8 3.3 91.7
200 78.1 16.3 55.2 63.0
Phase composition was 5 wt% PEG 8000, 14 wt% Reppal PES
200, 0.5 wt% IgG-Eudragit (60 mg of IgG/g of Eudragit) and 0.2M
K phosphate. Total weight of system was 5 g (0.025 g of IgG-
Eudragit).
E l : first fraction of eluate; E2: second fraction of eluate.
a Recovery was calculated from added amount and eluted amount.
1385
Purification of Recombinant Protein A
This separation method was applied for purification of
protein A produced by recombinant E. coli. The cell ho-
mogenate was directly added without elimination of cell
debris to the aqueous two-phase system containing IgG-
Eudragit. Because the cell debris was partitioned to the
bottom phase after phase separation, this method pro-
vided the advantage in terms of removal of cell debris
without the aid of a high-speed centrifuger, etc.
Table V shows the results of purification of the re-
combinant protein A from the cell homogenate. Two
different sample amounts were used for purification.
Addition of sample containing 137 pg protein A (total
soluble protein was 4400 pg) resulted in partitioning of
95% of the protein to the top phase, and 85% of this
could be recovered after the precipitation of IgG-Eu-
dragit. As a result, the total recovery was 80.3%. Most
Figure 4. SDS-PAGE patterns for purified protein A. Lane M:
of the protein A was recovered in the first fraction of marker proteins. Lane 1: crude extract without cell debris. Lane 2:
eluate. The purity of the first fraction was 81% and en- first fraction of eluate purified by aqueous two-phase extraction
hanced 26-fold as compared with that of the crude ex- with affinity precipitation. Lane 3: eluate from IgG-sepharose.
tract without cell debris.
Figure 4 shows SDS-PAGE patterns of purified pro- IgG-Eudragit added was lost each time due to incom-
tein A samples. The main band of the first fraction of plete recovery of the top phase, the recovery of protein
the eluate appeared in the same position (MW 31,000) A adsorbed to IgG-Eudragit was maintained at around
as the protein A purified from the same cell by IgG- 90% for every cycle (data not shown). The purity of the
sepharose. However, the purity of the second fraction of eluates during repeated use was 35% to 50% (data not
eluate was lower than that of the first fraction. SDS- shown). Relative adsorption capacity and total protein
PAGE analysis revealed that this finding was not due to on IgG-Eudragit, including immobilized IgG after each
denaturation of protein A, but due to elution of impuri- cycle, are shown in Figure 5. As seen, the relative ad-
ties nonspecifically bound to IgG-Eudragit (data not sorption capacity was decreased to 80% in the first use,
shown). and thereafter was maintained constant. The reason for
When the amount of the protein A sample added to the decrease in adsorption capacity seemed to be the
the two-phase system was doubled, the total recovery denaturation of the ligand and/or some protein A still
was decreased to 55.3%, and the purity of the first frac- remaining on IgG-Eudragit due to incomplete elution.
tion of eluate was also lower (Table V). Therefore, over- On the other hand, proteins were accumulated on IgG-
load is not recommended in this procedure in terms of Eudragit in the use. After five cycles of purification,
both the loss of product and the decreased purity. the total protein amount on Eudragit became 224 mg/g
Next, the repeated use of IgG-Eudragit in the two- Eudragit, and the accumulated amount was 2.7-fold
phase system was examined. Although 3% to 5% of higher than the amount of immobilized IgG. It is as-
sumed, therefore, that the adsorbed proteins caused the
Table V. Purification of recombinant protein A from cell homo- decrease in the purity of eluate during repeated use.
genate by the PEG 8000-Reppal PES 200 two-phase system con-
taining IgG-Eudragit.
100 (h I_ 500
Added Recovered protein A (pg)
protein A Recovery a
(f%) El E2 Bottom (%I
137 80.0 30.1 6.7 80.3
(0.031)b (0.810)b (0.494)b
274 112.0 39.8 53.0 55.3
(0.031)b (0.563)b (0.519)b

Phase composition was 5 wt% PEG 8000, 14 wt% Reppal PES

200, 0.5 wt% IgG-Eudragit (60 mg of IgG/g of Eudragit) and 0.2M 0 1 2 3 4 5 6
K phosphate. Total weight of system was 5 g (0.025 g of IgG-
Number
Eudragit).
El: first fraction of eluate; E2: second fraction of eluate. Figure 5. Relative adsorption capacity and total protein amount
a Recovery was calculated from added amount and eluted amount. on repeatedly used IgG-Eudragit. Symbols: 0, relative adsorption
Protein A content (mg of protein A/mg of total protein). capacity; 0, total protein on Eudragit.

1386 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 40, NO. 11, DECEMBER 20, 1992
Nevertheless, IgG-Eudragit could be repeatedly used
for separation of protein A. Further studies concerned
with washing conditions or regeneration method of IgG-
Eudragit will be necessary to prevent the decrease of
purity during reuse.
CONCLUDING REMARKS
Downstream processing is often characterized by a se-
quence of several separation steps that all contribute to
reach a product with the desired purity. Losses in each
step lead to reduction in yield in the overall process.
One way to improve the yields would be to reduce the
number of steps. This could be achieved by introducing
a step with selectivity and capacity and/or by integrating
different unit operations into one new separation step.
The present report deals with the combination of two
techniques, each of which holds great potential in down-
stream processing, but is also characterized by certain
limitations, i.e., extraction in aqueous two-phase sys-
tems and precipitation of affinity complexes. The idea
behind this integration being to combine the best of
both these techniques, i.e., removal of cell debris by af-
finity extraction in aqueous two-phase systems and af-
finity precipitation from the top phase. This latter part
facilitates the recovery of the target protein from all the
phase polymer that needs to be separated before a pure
product is achieved. Efforts in the same direciton have
been presented b e f ~ r e . ~ " ~In" those cases, the tech-
niques were developed suitable for the laboratory-scale
situation, but the present report deals with a technique
that consists of unit operations well known to operate
efficiently on a large scale.
The separation method by aqueous two-phase system
with affinity precipitation described in this study should
be applicable for industrial scale purification as a new
purification procedure. Although it may be possible to
isolate the protein simply by affinity precipitation, com-
bination with the aqueous two-phase system helps in the
isolation of the protein from cell debris and other
macromolecular substances and, hence, has the feasibil-
ity of being directly integrated with the bioreactor. This
purification method can be used for other affinity pro-
cesses, such as dye affinity and antigen-antibody, al-
though it is essential to select proper aqueous two-phase
system and processing conditions according to the affin-
ity system under study.
The authors thank the Japan-Sweden Foundation, Tokyo,
Japan; National Swedish Board for Industrial and Technical
Development (NUTEK); and the Swedish Research Council
for Engineering Sciences (TFR) for financial assistance.
KAMIHIRA, KAUL, AND MATTIASSON: AQUEOUS TWO-PHASE WITH AFFINITY PRECIPITATION
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