Protein Expression and Purication 54 (2007) 110116

www.elsevier.com/locate/yprep

The eects of arginine on protein binding and elution

in hydrophobic interaction and ion-exchange chromatography
Tsutomu Arakawa a, Kouhei Tsumoto b, Kazuo Nagase c, Daisuke Ejima
b

c,*

a
Alliance Protein Laboratories, Thousand Oaks, CA 91360, USA
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba-ku, Sendai 980-8579, Japan
c
Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan

Received 24 December 2006, and in revised form 15 February 2007

Available online 27 February 2007

Abstract
Arginine is eective in suppressing aggregation of proteins and may be benecial to be included during purication processes. We
have shown that arginine reduces non-specic protein binding in gel permeation chromatography and facilitates elution of antibodies
from Protein-A columns. Here we have examined the eects of arginine on binding and elution of the proteins during hydrophobic interaction (HIC) and ion-exchange chromatographies (IEC) using recombinant monoclonal antibodies (mAbs) and human interleukin-6. In
the case of HIC, the proteins were bound to a phenyl-Sepharose column in the presence of ammonium sulfate (AS) with or without arginine and eluted with a descending concentration of AS. While use of 1 M AS in the loading buer resulted in complete binding of the
mAb, inclusion of 1 M arginine in loading and equilibration buer, only when using low-substituted phenyl-Sepharose, resulted in
weaker binding of the proteins. While decreasing AS concentration to 0.75 M resulted in partial elution of the mAB, elution was facilitated with inclusion of 0.51 M arginine. In the case of IEC, arginine was included in the loading samples. Inclusion of arginine during
binding to the IEC columns resulted in a greater recovery and less aggregation even when elution was done in the absence of arginine.
These results indicate that arginine enhances elution of proteins bound to the resin, suggesting its eectiveness as a solvent for elution in
HIC and IEC.
 2007 Elsevier Inc. All rights reserved.
Keywords: Arginine; Hydrophobic interaction chromatography; Ion-exchange chromatography; Elution; Antibody; Ammonium sulfate

We and other groups have shown that arginine is eective in suppressing aggregation of proteins [1,2] and hence
may be benecial to be included at moderate concentrations during column chromatography. It is critical therefore to understand the impact arginine may have on
performance of various chromatographies. We have shown
that arginine has a positive impact on Protein-A [3,4], gel
permeation [5] and dye-anity chromatographies [6]; i.e.,
arginine facilitated elution of antibodies from Protein-A
under mild pH, reduced non-specic protein binding in
gel permeation chromatography and increased the recovery
in dye-anity chromatography. Here we have examined

the eects of arginine on protein binding and elution in

hydrophobic interaction (HIC)1 and ion-exchange chromatography (IEC), which constitute the important analytical
and preparative separation systems for proteins [7,8].
HIC exploits the weak hydrophobic surface of both native
proteins and column matrix and hence requires strong salting-out solvents for binding [914]. In general, proteins are
bound to HIC columns at high ammonium sulfate (AS)
concentration followed by elution with a descending concentration of AS. Although AS is primarily used for
HIC, other salts, including glutamic acid (Na salt), sodium

Corresponding author. Fax: +81 44 244 5809.

E-mail address: daisuke_Ejima@ajinomoto.com (D. Ejima).

1046-5928/$ - see front matter  2007 Elsevier Inc. All rights reserved.
doi:10.1016/j.pep.2007.02.010

Abbreviations used: HIC, hydrophobic interaction chromatography;

IEC, ion exchange chromatography; mAb, monoclonal antibody; AS,
ammonium sulfate; IL-6, interleukin-6; AcONa, sodium acetate.

T. Arakawa et al. / Protein Expression and Purication 54 (2007) 110116

sulfate, guanidinium sulfate, aspartic acid (Na salt) and

NaCl, have also been used [15,16]. The most commonly
used salt for elution in IEC is NaCl, although other salts
can be used to modulate the ionic strength.
Arginine is also eective in solubilizing proteins from
insoluble pellets [17,18], increases the solubility of the proteins [19] and assists in protein refolding [2022]. In general, arginine is removed prior to the subsequent
purication steps. If arginine does not interfere with the
protein binding in the subsequent steps, then it can simply
be removed upon elution, which makes an extra step of
arginine removal unnecessary. Purication techniques,
which work in the presence of arginine, are desired. The
present results show that the proteins to be puried do bind
to the HIC and IEC columns in the presence of arginine
and the bound proteins can be eluted either with or without
arginine. In addition, it is demonstrated that inclusion of
arginine increases the recovery of the proteins from the columns and results in less aggregation.
Materials and methods
Materials
Recombinant human interleukin-6 (IL-6) was prepared
according to the methods reported previously [23]. Two
humanized monoclonal antibodies, IgG4-A and IgG4-B,
and a mouse monoclonal antibody, IgG1, were prepared
in Ajinomoto Co. All reagents are of biochemical research
grade. Arginine stock solution was prepared from L-arginine hydrochloride.
Methods
Hydrophobic interaction chromatography: A 1 ml
HiTrap low-substituted and high-substituted phenylSepharose columns (Amersham Biosciences) were equilibrated at a ow rate of 0.5 or 1 ml/min with an appropriate
buer containing AS in 10 mM phosphate. In some cases,
arginine was included in the equilibration buer and loading sample to mimic the condition at which proteins to be
puried are exposed to arginine in the prior step of protein
purication process. An appropriate volume of protein
samples, 2 M AS and 2 M arginine were mixed to generate
loading samples as indicated in each experiment. In all
experiments, 4 mg of the proteins were loaded on the column. Column chromatographies were carried out at 4 C.
The amount of the proteins eluted in each elution condition
was spectrophotometrically determined and expressed as a
percentage to the amount of total proteins loaded.
Ion-exchange chromatography: Following the previous
observation [23] that inclusion of sodium acetate in the
loading sample increases the recovery of monomeric IL-6,
the eects of replacing sodium acetate with arginine or
NaCl were examined. The HiTrap CM-Sepharose IEC column (1.6 10 cm) was equilibrated with 10 mM sodium
acetate, pH 5.8. After loading the IL-6 sample in an appro-

111

priate solvent (as indicated in the gures), the column was

eluted with a linear gradient of sodium acetate. For mAb, it
is customary to have IEC chromatography following Protein-A step and hence the IEC was done after elution of
the mAb from the Protein-A chromatography process.
The mAb was bound to the 1 ml HiTrap Protein-A column
and eluted with 0.5 M arginine at pH 4. Then the conditions for binding of the Protein-A pool to the IEC column
and subsequent elution from the column were examined.
Results
Hydrophobic interaction chromatography
In the rst experiment, 4 mg of IgG4-A were loaded
to the phenyl-Sepharose (low-substituted) in 1 M AS,
10 mM phosphate, pH 6.0, which resulted in complete
binding. Elution was initiated by decreasing AS concentration to 0.75 M AS in 10 mM phosphate, pH 6.0, resulting
in elution of only a fraction of the protein applied (16%,
rst column) and the remaining bound proteins eluting
with lower AS concentrations, as summarized in Fig. 1.
It is evident that 0.75 M AS is insucient for eective elution of the bound IgG4-A. Next, 1 M arginine was
included in the 0.75 M AS elution buer, resulting in much
higher recovery (45%, second column of Fig. 1). In these
experiments, arginine was not included in the equilibration
buer and loading sample. In order to mimic the condition
of arginine-containing samples [11,12], the following experiments were carried out to load the same mAb in the presence of arginine. First, the HIC column was equilibrated
with 1 M AS/0.5 M arginine and then the mAb in 1 M
AS/0.5 M arginine loaded. Under this condition, a small
amount of the protein owed-through the column (data
not shown), indicating that 0.5 M arginine weakens

70
60
50
40
30
20
10
0
None

1 M Arg

Loading without arginine

0.5 M Arg

1 M Arg

1 M Arg

Loading with arginine

Fig. 1. Elution of IgG4-A from low-substituted phenyl-Sepharose using

0.75 M AS with and without arginine. In all cases, 4 mg IgG4-A were
loaded in the presence of 1 M AS. Although the elution conditions
were identical for the fourth and fth columns, binding conditions were
dierent, i.e., 1 M AS/1 M Arg for the fourth column and 1 M AS/0.5 M
Arg for the fth column.

112

T. Arakawa et al. / Protein Expression and Purication 54 (2007) 110116

60

Total recovery yield

hydrophobic interaction of the protein with the HIC column. After washing the column with 1 M AS/0.5 M arginine, the bound protein was eluted with 0.75 M AS/
0.5 M arginine (third column of Fig. 1). The elution recovery with the 0.75 M AS/0.5 M arginine was intermediate
(30%) between 0.75 M AS alone (rst column) and
0.75 M AS/1 M arginine (second column), suggesting that
the ability of arginine to dissociate proteins from ProteinA is concentration-dependent. Arginine concentration for
loading was increased to 1 M arginine; i.e., the column
was equilibrated with 1 M AS/1 M arginine and the mAb
in the same buer was loaded. The ow-through peak
increased compared to that observed with 1 M AS/0.5 M
arginine (data not shown), indicating that an increase in
arginine concentration to 1 M further weakened binding
of the mAb to the HIC column. Elution was more ecient
with 0.75 M AS/1 M arginine than with 0.75 M AS/0.5 M
arginine (52%, fourth column). Based on the above experiments, 0.5 M arginine (and probably less arginine) is more
optimal for binding, while elution is ecient with 1 M arginine (and probably higher arginine concentrations). Therefore, an experiment shown in the fth column was carried
out, in which the mAb was bound in 1 M AS/0.5 M arginine to the HIC column equilibrated with the same buer.
The ow-through peak decreased compared to that with
1 M AS/1 M arginine, consistent with the experiment of
the third column. A sharp elution peak was observed with
0.75 M AS/1 M arginine with a recovery of 66%. Based on
these observations, one should adjust the arginine concentration of the loading sample to an optimal level for complete binding and achieve elution by an appropriate
concentrations of AS and arginine.
Such a concern appears unnecessary when using highsubstituted phenyl-Sepharose. Binding was complete even
in the presence of 1 M arginine (data not shown), consis-

50

tent with higher ligand density and hence more hydrophobic surface of this resin. When the IgG4-A was bound in
1 M AS without arginine and bound IgG4-A eluted with
0.5 M AS alone, the recovery was only 4% from this column (rst column, Fig. 2), consistent with the more hydrophobic surface. The elution pattern was also extremely
broad, indicating that 0.5 M AS is insucient for elution.
However, elution increased by the addition of 0.5 and
1 M arginine to the 0.5 M AS elution buer. Namely, the
same mAb was bound to the column in 1 M AS and then
bound protein eluted with 0.5 M AS containing 0.5 or
1 M arginine, which resulted in 25 and 55% elution (second
and third column). A combination of 0.5 M AS with 1 M
arginine for elution resulted in a sharp peak (see Fig. 2, last
prole). Elution recovery further increased when the loading sample also contained 1 M arginine or the bound IgG4A was eluted with 10 mM phosphate alone (i.e., without
AS). The recovery was 50% with 10 mM phosphate alone
and increased nearly to 100% when the 10 mM phosphate
elution buer contained 0.5 or 1 M arginine (data not
shown).
Essentially identical results were obtained with mouse
IgG1. As shown in Fig. 3, mouse IgG1 was loaded to the
high-substituted phenyl-Sepharose in 1 M AS, 10 mM
phosphate, pH 7.0 containing 1 M arginine, resulting in
complete binding and hence again demonstrating that
inclusion of arginine even at such high concentration does
not interfere with binding. About 60% of the loaded material was eluted using 10 mM phosphate. The elution recovery increased to >80% when the elution buer contained
0.5 and 1 M arginine. In this case, 0.5 M arginine appears
to be sucient for higher recovery. These results demonstrate that arginine has little impact on protein binding to
HIC columns, in particular using the high-substituted phenyl-Sepharose.

40
30
20
10
0

1 M AS

Elution

0.5 M AS

1 M AS

1 M AS

0.5 M AS + 0.5 M A rg
0.5 M AS + 1.0 M A rg

Abs280

Loading

Elution
Fig. 2. Elution of IgG4-A from high-substituted phenyl-Sepharose using 0.5 M AS with and without arginine. Due to the limited space, arginine is
abbreviated as Arg in this and the in Fig. 3.

T. Arakawa et al. / Protein Expression and Purication 54 (2007) 110116

100
80
60
40
20
0

Loading

1 M AS + 1 M Arg

Elution

10 mM phosphate

1 M AS + 1 M

1 M AS + 1 M Arg

10 mM phosphate + 0.5 M Arg 10 mM phosphate + 1.0 M Arg

Fig. 3. Elution of mouse IgG1 from high-substituted phenyl-Sepharose

using 10 mM phosphate with and without arginine.

Ion-exchange chromatography
Previously we have observed that inclusion of sodium
acetate in the loading sample increased the recovery of
IL-6 and decreased its aggregation during IEC [23]. We
have thus examined the eects of arginine using the same
protocol. IL-6 was bound to the CM-Sepharose in the
absence and presence of 0.2 M salts in 20 mM sodium acetate (AcONa) and the bound protein eluted by raising the
AcONa concentration to 0.5 M. Binding of IL-6 was complete even in the presence of 0.2 M arginine, indicating no
interference with the binding. The elution results are summarized in Fig. 4. As has been observed before, the addition of 0.2 M sodium acetate increased the recovery by
8% (from 80 to 88%) and decreased aggregation, as determined by analytical size exclusion chromatography, by
13% (from 21 to 8%), as seen in Fig. 4 (compare rst and
last column). An increase in recovery (to 82%) and a
decrease in aggregation (to 10%) was observed with
0.2 M NaCl (Fig. 4, third column). Recovery further
increased to 90% when the sample loading was done in
the presence of 0.2 M arginine (second column). More
importantly the amount of aggregates decreased to less

100

50
45

80

40
35

60

30
25

40

20
15

20

10
5

0
No additive

0.2 M
Arg

0.2 M
NaCl

Additive (M)

0.2 M
AcONa
Total recovery yield
Aggregate content

Fig. 4. Elution of IL-6 from CM-Sepharose. IL-6 was loaded with and
without 0.2 M salts.

113

than 1% using 0.2 M arginine. Here not only arginine

was compatible with protein binding to the IEC column,
but its inclusion in the loading sample reduced aggregation.
This reduction may be due to suppression of protein-protein interaction during loading of IL-6.
Binding of IL-6 was not aected by the presence of
0.2 M arginine. Does arginine alter the elution when present in the elution solvent? This was tested by a salt gradient
elution of IL-6 from the CM-Sepharose at pH 5.0. When
eluted with NaCl alone, the protein elution consistently
occurred at 0.375 M salt. When both the starting and ending elution solvents contained 0.2 M arginine, the protein
elution occurred at 0.2 M NaCl. Thus, it is evident that
arginine does contribute to elution as an ion, mostly as a
monovalent ion at pH 5.0. The slightly higher total salt
concentration, i.e., 0.4 M, required for elution suggests that
the eectiveness of arginine as an ion is weaker than NaCl,
which may be due to its larger size.
A platform approach has been developed for purication of mAbs [24], in which IEC follows the Protein-A step
in the down-stream process. We have shown that an aqueous arginine solution facilitates the elution of bound antibodies from Protein-A at higher pH, reducing deleterious
eects of low pH on the antibody structure and subsequent
aggregation. It is thus desirable that the eluted antibody be
bound to the next column, i.e., IEC, without removing
arginine. After searching for the condition for IEC, we
found that 0.25 M arginine is acceptable for binding mAbs
to SP-Sepharose at pH 4.2. Table 1 summarizes the recovery and aggregate content of IEC process following the elution from Protein-A. The human IgG-4A was loaded to
Protein-A column at 20 mg protein per ml resin and eluted
with 0.1 M citrate, pH 2.9 (rst row of Table 1) or 0.5
0.7 M arginine at pH 4 (second to fth row); note that
duplicate experimental data are shown with similar results.
When using 0.1 M citrate, such a low pH was necessary for
higher elution of this particular mAb from Protein-A; in
fact, 8396% recovery was observed (see fourth column).
When the eluted material was subjected to a size exclusion
chromatography (SEC) analysis at neutral pH, it showed
3843% aggregates of the total eluted protein. As previously shown, the observed aggregation is most likely due
to low pH-induced conformational changes, which leads
to aggregation upon pH titration prior to the SEC analysis.
While the solvent pH was 2.9, the eluted pool was at pH
3.1, due to strong buer action of eluted antibodies.
Conversely, the recovery of eluted protein is close to
100% when using arginine at much higher pH (e.g., 0.5
0.7 M arginine at pH 4). The pH of the eluted pools
was slightly above the pH of the elution solvents, similar
to the previous observation. In addition to high recovery
using arginine, there is little aggregation as analyzed by
SEC (below 1% as seen experiment 25 in Table 1). The
advantage of 0.1 M citrate is that the eluent can be applied
directly to the next IEC column. However, the recovery
from the IEC column was also low and the aggregate
content was high. The overall yield was 5774% and the

114

Table 1
Elution of IgG-4A from Protein-A and SP-Sepharose
Experiment
No.

Eluent

Protein-A
pH of Total
collected protein
fraction (%)

Aggregate
content
(%)

Buer condition
of loading sample

Eluent

Total protein
(%)

Aggregate Total
content
protein
(%)
(%)

Monomer
(%)

Relative
yield
of monomer

11
12

0.1 M
citrate (pH 2.9)

3.13
3.10

83.1
96.4

43.3
38.4

0.1 M citrate
(pH 3.5)

0.1 M citrate + 0.25 M

NaCl (pH 4.0)

68.7
76.4

11
33

57.1
73.6

50.9
49.3

21
22

0.5 M Arg
(pH 3.9)

4.13
4.20

92.6
100

0.5
0.9

0.25 M Arg + 20 mM
AcONa (pH 4.0)

0.5 M AcONa
(pH 5.5)

84.9
92.5

1.1
1.2

78.6
92.5

77.7
91.4

1.55
1.82

31
32

0.7 M Arg
(pH 3.9)

4.11
4.17

95.5
100

0.9
0.8

0.2 M Arg + 20 mM
AcONa (pH 4.2)

0.5 M AcONa
(pH 5.5)

87.9
93.0

1.1
1.4

83.9
93.0

83.0
91.7

1.66
1.83

41
42

0.7 M Arg + 50 mM
AcONa (pH 4.0)

4.10
4.14

100
96.0

0.7
0.8

0.2 M Arg + 20 mM
AcONa (pH 4.2)

0.5 M AcONa
(pH 5.5)

92.4
91.4

1.3
1.3

92.4
87.7

91.2
86.6

1.82
1.73

51
52

0.5 M Arg
(pH 3.8)

4.06
4.05

98.9
95.7

1.1
0.8

0.25 M Arg + 20 mM
AcONa (pH 4.0)

0.5 M AcONa
(pH 5.5)

85.7
96.7

1.6
1.3

84.8
92.5

83.4
91.3

1.67
1.82

Cation exchange

Total yield

Experiment Protein A
No.
Eluent

Cation exchange

Total yield

pH of collected
fraction

Total
protein
(%)

Aggregate
content
(%)

Buer condition
of loading sample

Eluent

Total
protein
(%)

Aggregate
content
(%)

Total
protein
(%)

Monomer Relative
(%)
yield of
monomer

0.1 M citrate + 0.25 M

NaCl (pH 4.0)
0.5 M AcONa (pH 5.5)

40.2

21.1

36.6

28.9

84.8

1.7

76.2

74.9

2.59

0.5 M AcONa (pH 5.5)

85.6

1.7

82.0

80.6

2.79

11

0.1 M citrate (pH 2.9)

3.13

91.0

24.0

0.1 M citrate (pH 3.5)

21

0.5 M Arg (pH 3.9)

4.15

89.8

1.0

31

0.7 M Arg + 50 mM
AcONa (pH 4.0)

4.09

95.8

1.0

0.25 M Arg + 20 mM
AcONa (pH 4.0)
0.2 M Arg + 20 mM
AcONa (pH 4.2)

T. Arakawa et al. / Protein Expression and Purication 54 (2007) 110116

Table 2
Elution of IgG-4B from Protein-A and SP-Sepharose

T. Arakawa et al. / Protein Expression and Purication 54 (2007) 110116

monomer recovery was only 50%. The aggregate content

after the IEC was 1133%. The observed lower aggregation
content of one case (11%) may be due to the lower protein
recovery (69%), after SP-Sepharose, due to preferential loss
of aggregates in the column.
Use of arginine to elute from Protein-A may be disadvantage, since at least 0.5 M is required for ecient elution
above pH 4. Dilution to 0.25 M arginine was, however,
found sucient for binding to SP-Sepharose at pH 4.0
4.2. Elution of the bound antibody from the IEC column
was always 90%, leading to the overall recovery of
8095% with the monomer recovery of 8090%. As
shown in the Table 1, the aggregate content was much less
with arginine elution (slightly above 1% after IEC column)
than citrate elution. A shown in the last column, the overall
recovery of the monomer exceeds 150% when using arginine in the Protein-A step, relative to the recovery without
arginine.
The IEC experiment was also carried out with IgG4-B.
About 90% of the antibody was recovered from ProteinA using 0.1 M citrate, pH 2.9, which generated 24% aggregates during SEC analysis (rst row of Table 2). This was
loaded to SP-Sepharose and eluted using 0.1 M citrate,
0.25 M NaCl, pH 4.0 with the recovery of only 40%. The
overall yield was only 36% and the monomer recovery
was 29% with aggregate content of 21%. When eluted with
0.50.7 M arginine, pH 4, from Protein-A, the elution
recovery was similar, but with much less aggregation
(1%)(second and third row). This was diluted 2-fold and
then loaded to the same column at pH 4. Elution from
the column resulted in the recovery of 85% and aggregate
content of 1.7% after SP-Sepharose. In this case, the monomer content in the nal product was greater than 250%
(last column of Table 2). The observed lower amounts of
aggregates in the nal product using arginine is due to less
damage in the Protein-A eluent; in other words, whatever
the damage suered in the Protein-A step, that is responsible for aggregation, is carried over to the nal product.
This means that inclusion of arginine in the SP-Sepharose
step does not, positively or negatively, aect the nal product of the mAbs tested here.
Discussion
The goal of this study was to examine the eects of arginine on protein binding to the HIC and IEC columns. It is
evident that the proteins tested do bind to these columns in
the presence of arginine, when its concentration during
protein binding is optimized and an appropriate column
is selected. We and others have shown that arginine facilitates refolding [1922], suppresses aggregation [1,2],
increases reversibility of thermal unfolding [1,2], solubilizes
insoluble pellets [17,18], dissociates antibodies or Fc-fusion
proteins from Protein-A [3,4] and reduces non-specic
binding of proteins, in particular aggregates, during gel
permeation chromatography [5]. Here we demonstrated
that arginine weakens hydrophobic interactions and facili-

115

tates elution of bound proteins from phenyl-Sepharose

during descending AS concentration. In addition, inclusion
of arginine in the loading sample increased the recovery of
the total protein and decreased the aggregation during
IEC.
AS has been the salt most commonly used for modulating binding and elution of proteins in HIC. Why is
AS the salt of choice? Extending an earlier observation
by Hofmeister and Traube on salting-out or salting-in
eects of salts, Melander and Horvath [25] showed that
the surface tension increments of salts in water correlate
with the ability of the salts to enhance protein binding
to HIC columns, with the exception of certain salts, such
as MgCl2. Preferential interaction measurements conrmed the importance of surface tension eects and
showed why certain salts deviate [2628]. Based on the
surface tension eects and preferential interactions with
the proteins, AS is considered to be the salt of choice
due to its high solubility. It is also evident from the surface tension and preferential interactions that salts, other
than AS, can be used for HIC. In fact, other additives,
although limited, have been used to modulate binding
and elution in HIC. Here we have shown that not only
HIC can be performed in the presence of arginine, but
also arginine increases the recovery of the proteins.
We have also shown here that both mAb and IL-6 can
be bound to the IEC columns in the presence of arginine.
In addition, inclusion of arginine in the loading samples
enhanced elution of IL-6 and reduced its aggregation.
How does arginine work in IEC and HIC? Arginine has
been demonstrated to suppress aggregation [1,2] and nonspecic surface adsorption of the proteins [5]. It is thus possible that arginine also suppresses non-specic binding of
IL-6 and mAb to the HIC and IEC columns and reduces
aggregation of IL-6 during binding to the IEC column.
What is the mechanism of arginine in suppressing protein
aggregation and non-specic binding? Arginine has been
implicated to bind, although to limited extent, to the proteins [29,30]. GdnHCl, which contains a guanidinium
group present in arginine, has high anity for aromatic
groups [31] and interacts with tryptophan side chains
[32]. It thus appears that a limited extent of arginine binding is responsible for its eect on suppressing aggregation
and non-specic adsorption of the proteins.
These observations open a way one can use for purifying
proteins after solubilization or elution by arginine-containing solvents without its removal. Since arginine solubilizes
certain proteins from insoluble pellets in the active, folded
structure [17,18], HIC in the presence of arginine should be
a useful technique for purication of the proteins without
potential denaturation. Arginine facilitates elution of antibodies from Protein-A under mildly acidic pH. Here we
have shown that HIC and IEC can be used without removing arginine as a next step to Protein-A chromatography.
Of course, it would be advantageous if more chromatography options, applicable to the arginine-containing solvent,
are available.

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T. Arakawa et al. / Protein Expression and Purication 54 (2007) 110116

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