Effects of Tween 20

1
and Tween 80
1
on the Stability
of Albutropin During Agitation
DANNY K. CHOU,
1
RAJESH KRISHNAMURTHY,
3
THEODORE W. RANDOLPH,
2
JOHN F. CARPENTER,
1
MARK CORNELL MANNING
1
1
Department of Pharmaceutical Sciences, School of Pharmacy, Center for Pharmaceutical Biotechnology,
University of Colorado Health Sciences Center, Denver, Colorado 80262
2
Department of Chemical Engineering, Center for Pharmaceutical Biotechnology, ECCH 111, Campus Box 424,
University of Colorado, Boulder, Colorado 80309
3
Human Genome Sciences, Rockville, Maryland 20850
Received 2 November 2004; revised 2 March 2005; accepted 13 March 2005
Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20365
ABSTRACT: The objectives of this work were to determine the effects of nonionic
surfactants (Tween 20
1
and Tween 80
1
) on agitation-induced aggregation of the
recombinant fusion protein, Albutropin
TM
(human growth hormone genetically fused to
human albumin), and to characterize the binding interactions between the surfactants
and the protein. Knowing the binding stoichiometry would allow a rational choice of
surfactant concentration to protect the protein from surface-induced aggregation.
Fluorescence spectroscopy and isothermal titration calorimetry (ITC) were employed to
study Albutropin surfactant binding. Albutropin was agitated at 25 28C to induce
aggregation, and samples were taken during a 96-h incubation. Size-exclusion chroma-
tography (SEC-HPLC) (HPLC, high-performance liquid chromatography) was used to
detect and quantify the extent of protein aggregation. The effect of surfactants on the
proteins free energy of unfolding was determined using guanidine HCl as a denaturant.
Tween 20 and Tween 80 had saturable binding to Albutropin with a molar binding
stoichiometryof 10:1and9:1(surfactant:protein), respectively. Bindingof thesurfactants
to Albutropin increased the free energy of unfolding by over 1 and 0.6 kcal/mol,
respectively. In protein samples that were agitated in the absence of surfactant, soluble
aggregates were detected within 24 h, and there was almost complete loss of monomer to
soluble aggregates by the endof the 96-hexperiment. At the molar binding stoichiometry,
Tween 20 and Tween 80 prevented the formation of soluble aggregates, even though the
concentrations of surfactants were well below their critical micelle concentrations
(CMC). Tween 20 and Tween 80 protected Albutropin against agitation-induced aggre-
gation, even at concentrations belowthe CMC. Equilibrium unfolding data indicate that
Tween confer protection by increasing the free energy of unfolding of Albutropin. 2005
Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 94:13681381, 2005
Keywords: protein aggregation; Tween 20; Tween 80; nonionic surfactant; uore-
scence; isothermal titration calorimetry
INTRODUCTION
Because most pharmaceutical proteins are sur-
face-active and only marginally stable, interac-
tions with interfaces (e.g., air-water and solid-
water interfaces) often lead to loss of native
structure and aggregation.
1
Aggregation can
compromise biological activity and/or induce an
1368 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
Mark Cornell Mannings present address is Legacy BioDe-
sign LLC, Loveland, CO 80538.
Correspondence to: Mark Cornell Manning (Telephone: 970-
231-9744; Fax: 970-663-6006.;
E-mail: legacybiodesign@cs.com)
Journal of Pharmaceutical Sciences, Vol. 94, 13681381 (2005)
2005 Wiley-Liss, Inc. and the American Pharmacists Association
immunological reaction in the patient.
2,3
Thus,
the formulation must be designed to protect the
protein against surface-induced damage. Surfac-
tants, particularly nonionic types, are often added
to prevent and/or minimize protein aggregation
during fermentation, purication, freeze-drying,
shipping, and/or storage.
4
There are several known mechanisms for pro-
tein stabilization by surfactants. First, nonionic
surfactants can protect proteins against surface-
induced damage by competing with proteins for
adsorption sites on surfaces.
4
In addition, as has
been demonstrated with human growth hormone
(hGH), nonionic surfactants can protect protein
against surface-induced aggregation by binding to
hydrophobic regions of the surface of the protein
molecule, and thus decrease intermolecular in-
teractions.
5,6
In these cases, the concentration of
surfactant required to protect the protein maxi-
mally fromaggregation does not correlate with the
critical micelle concentration (CMC) of the sur-
factant. Rather, the degree of protection can be
maximized at the molar binding stoichiometry
between the surfactant and protein.
5,6
Binding of
surfactant to the native state of the protein can
also minimize surface-induced aggregation by
increasing the free energy of protein unfolding,
as has also been found with hGH.
7
Finally, nonio-
nic surfactants may act as a chemical chaperone,
favoring refolding over aggregation by binding
transiently withpartially folded protein molecules
and sterically hindering intermolecular interac-
tions that result in aggregation.
8,9
Currently, the most commonstrategy employed
for stabilizing protein pharmaceuticals against
surface-induced aggregation is to include in the
formulation a sufcient concentration of surfac-
tant to minimize protein adsorption to interfaces.
Typically, the nal surfactant concentrationis just
above the CMC, where there is a monolayer of
surfactant molecules oriented about the interface
(e.g., the air-water interface).
1012
Although this
approach can minimize the interaction of the
protein with denaturing interfaces (air-water,
solid-water interfaces, etc.), it does not take into
account the binding between the surfactant and
the protein. For example, studies have shown that
a protein can change the CMC of a surfactant by
binding to surfactant molecules.
5,13
Because one
goal of formulation development is to utilize the
lowest effective concentration of excipient, a sys-
tematic approach to surfactant formulation de-
velopment is needed. Through such an approach,
potential surfactant binding to the protein as well
as the competition between protein and surfactant
for surfaces can be evaluated.
Albutropin
TM
is a novel form of hGH, geneti-
cally fused to human serum albumin (HSA). Al-
butropin is expressed and puried to yield a single
polypeptide with a substantially longer half-life in
vivo than that for hGH alone.
14
Human serum
albumin is the most prevalent naturally occurring
protein in the human circulatory system, persist-
ing in circulation in the body for over 20 days.
15
Albumin fusion proteins may thus provide pa-
tients withlong-actingtreatment options that may
offer a more convenient dosing regimen, with
similar or improved efcacy and safety. To date,
there has not been published work on agitation-
induced aggregation of albumin fusion proteins,
nor on the effects of nonionic surfactants on this
process. Furthermore, although previous studies
involving surfactant formulation of hGH de-
monstrated the importance of a specic protein-
surfactant stoichiometry, the concentration of
surfactants utilized were well above their CMC.
6
Thus, another goal of this study is to determine
whether sub-CMC concentration of a surfactant is
sufcient to protect a protein against agitation-
induced aggregation.
HSA is a major transporter of fatty acids in the
blood stream, and the afnity of HSA for both
medium and long chain fatty acids is high (10
6

10
7
M
1
).
16,17
In fact, the organic fatty acid,
octanoic acid, is commonly added to the protein
to protect it from denaturation during heat treat-
ment of commercial HSA products.
16
Given this
behavior and the known binding of surfactants to
hGH, we hypothesize that nonionic surfactants
will bind to Albutropin as well. Using the informa-
tion gathered fromsurfactant binding studies, the
hypothesis that a sub-CMC amount of Tween 20
1
and Tween 80
1
is sufcient to protect Albutropin
maximally against agitation-induced aggregation
was tested. The requisite concentration of surfac-
tant should correspond to or exceed the binding
stoichiometry between the surfactant and the
protein. In addition, studies were conducted to
test the effects of the surfactants on the proteins
free energy of unfolding.
MATERIALS AND METHODS
Protein and Reagents
Puried Albutropin (88.6 kD), expressed in yeast
(Saccharomyces cerevisiae) was supplied by
Human Genome Sciences (Rockville, Maryland)
EFFECTS OF NONIONIC SURFACTANTS 1369
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
at pH 7.2 and was stored at 808C until needed.
After thawing and dialysis against 10 mM
sodium phosphate buffer, size-exclusion high-
performance liquid chromatography (SE-HPLC)
analysis documented that the protein sample
contained approximately 99% monomer and 1%
dimer. Sodium phosphate monobasic, sodium
phosphate dibasic, potassium phosphate mono-
basic, guanidine hydrochloride (GdnHCl), poly-
oxyethylene sorbitan monolaurate (Tween 20),
and polyoxyethylene sorbitan monooleate (Tween
80) (Sigma Ultra, low peroxide) were purchased
from Sigma Chemical Company. All protein and
excipient solutions were prepared using distil-
led deionized water. Buffer solutions were ltered
through 0.22-mm nylon lters.
Fluorescence Titration Studies
The protein concentration was determined on a
HP model 8452 spectrophotometer using an
extinction coefcient (e
280
) of 0.6 mL mg
1
cm
1
1 for Albutropin (data from HGSI). The e
280
were
0.81 and 0.58 mL mg
1
cm
1
for hGH and HSA,
respectively.
16,43
Measurements of steady-state
uorescence were performed on a 0.05 mg/mL pro-
tein solution (258C) in sodium phosphate buffer
(10 mM at pH 7.2), with excitation wavelength at
295 nm in an Aviv ATF 105 automatic titrating
spectrouorimeter (Lakewood, NJ). Excitation
bandwidth was set at 0.15 nm and emission band-
width was set at 8 nm. For titration of the protein
with surfactant, each injection of surfactant solu-
tion was 2.6 mL in volume and the sample was
kept at a constant volume of 2 mL throughout the
experiment and stirred with a magnetic stir bar.
After each injection, the sample was allowed to
equilibrate for 1 min prior to spectral acquisition.
The uorescence intensity of the protein was
monitored at the wavelength of maximum emis-
sion for each protein (340 nm for Albutropin,
339 nm for rhGH, and 345 nm for HSA). All uor-
escence experiments were run in duplicate,
intensity was corrected for dilution, and average
values are reported. Data were imported into
Microsoft Excel
TM
software for further proces-
sing, uorescence quenching data were analyzed
with the SternVolmer equation, and the effec-
tive quenching constant was derived from the
slope of the binding isotherm.
Isothermal Titration Calorimetry (ITC)
Microcalorimetry experiments were performed on
a Microcal, Inc. Omega titration calorimeter. Both
the ligand and protein solutions were ltered
through a 0.22-mm lter then degassed for at least
15 min using a magnetically stirred vacuum ap-
paratus. The injection syringe was lled with a
stock solution containing Tween 20 or Tween 80
at 10 mM. Twenty ve injections of 10 mL each
were made into a 1.3 mL calorimeter cell, which
contained either 10 mM phosphate buffer pH 7.2
or the same buffer containing 16 mg/mL of Al-
butropin. The cell was stirred at 375 rpm and was
thermally equilibrated at 258C for 3040 min
until a at baseline was obtained, then injections
were initiated. Analysis of protein samples after
completion of the titration by SEC documented
that the protein has not aggregated (data not
shown). The time between injections was set to
240 s. Control experiments were conducted by
titrating Tween into buffer solution and the re-
sulting heat of dilution was subtracted from the
actual binding isotherm prior to data analysis.
The resulting data were integrated using Origin
software from Microcal
1
to arrive at the enthalpy
generated for each Tween injection.
Operative CMC Determination Using
Surface Tensiometry
The effective CMC of both Tween 20 and Tween
80 solutions containing protein was determined
using a manually operated Fisher surface tensi-
ometer. This instrument employs the du Nou y
ring method which utilizes a platinum-iridium
test ring and two stainless-steel torsion wires.
The manufacturers manual denes the apparent
surface tension as the force necessary to separate
platinum-iridium ring from a liquid, and in this
case it is measured and read directly to 0.25 dyn/
cm.
18
Direct results were obtained with solutions
that contained increasing concentrations of sur-
factant, and the breakpoint in the surfactant
concentration versus surface tension curve is
the apparent critical concentration of surfactant
necessary to achieve the minimum surface ten-
sion, and thus corresponds to the effective CMC of
the surfactant.
19
SE-HPLC
SE-HPLC was utilized to quantify levels of mono-
meric protein and soluble aggregates. A Tosohaas
TSK 3000SWxL gel ltration column connected to
Beckman Gold chromatography system (Fuller-
ton, CA) was used. The mobile phase was 104 mM
sodium phosphate, 104 mM potassiumphosphate,
1370 CHOU ET AL.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
and 100 mM sodium sulfate (pH 6.7), and the ow
rate was 1.0 mL/min. At neutral pH, Albutropin
exists as a monomer with a molecular mass of
88.6 kDa. The amounts of native monomer, non-
native dimer, and larger soluble aggregates were
quantied using UV absorbance at a detection
wavelength of 280 nm. The samples, chromato-
graphy system, column, and buffer were all main-
tained at 258C throughout the course of the
experiment. Albutropin that had not been agi-
tated was used as a control. Data collected were
imported into Grams software program, and peak
areas were analyzed using Beckmans chromato-
gram integrator software.
Equilibrium Unfolding Experiments
Albutropin stock solution was dialyzed against
10 mM sodium phosphate buffer at pH 7.2 with
Pierce Slide-A-Lyzer dialysis cassette prior to
use. Guanidine hydrochloride (GdnHCl)-induced
unfolding of Albutropin at 0.1 mg/mL was con-
ducted with and without Tween 20 or Tween 80.
Stock solutions of 8M GdnHCl in buffer, with and
without Tween 20 or Tween 80 at concentrations
that correspond to stoichiometric molar ratio of
surfactant:protein, were prepared as described by
Pace et al.
20
Appropriate amounts of Tween 20
and Tween 80 solutions were added to the buffer
and protein solutions and then combined with
GdnHCl stock solutions to prepare protein sam-
ples with various GdnHCl concentrations. A total
of 30 samples with various concentrations of
GdnHCl, ranging from 0M to 6.4M were prepared
and equilibrated overnight at 48C prior to data
collection. All samples were performed in tripli-
cate and the results are the average of three
separate experiments. Denaturant induced un-
folding of Albutropin in 10 mM sodium phosphate
(pH 7.2) was followed using an Aviv circular
Dichroism (CD) spectrometer (model 62 DS). A
1-mm pathlength sample cell was used for all CD
spectroscopic measurements. Averages (60 s) of
far UV CD signals at 222 nm were collected.
Samples without Albutropin were used as blanks
to correct for non-Albutropin contributions to far
UV CD signal. The unfolding curves were ana-
lyzed using the method devised by Hung and
Chang and it is briey described below.
21
In a
three-state unfolding transition, the equilibrium
between the states can be described by the fol-
lowing equation:
N !

KN !I
I !

KI !D
D 1
Assuming linear free energy changes theory,
which states that each unfolding step has the
following relationship of the standard free energy
changes:
DG
0
i
DG
i
m
i
D 2
in which DG
i
is the free energy change of each
step, m represents the dependence of the DG on
denaturant concentration [D]. The following rela-
tionship holds true in terms of equilibrium con-
stants between each state
K
i
exp
DG
0
i
m
i
D
RT

3
in which R is gas constant and T is the absolute
temperature in kelvin. The method for analysis of
three-state transition unfolding curves is shown
in Equation 4.
Albutropin unfolding curves were tted to this
model with Igor Pro software using Equation 4,
and the results were chosen based on a combina-
tion of parameters that achieved the best t to the
data.
Agitation Studies
Triplicate samples containing 0.3 mg/mL Albu-
tropin (1 mL) of each formulation were placed into
2.0 mL polypropylene microcentrifuge tubes (dia-
meter 10 mm), which provided sufcient head
space for bubble entrainment into the solution
from the air-liquid interface during agitation.
Albutropin was formulated without surfactant
and with Tween 20 or Tween 80 slightly above the
critical micelle concentration (CMC) of each sur-
factant, at an amount of Tween 20 or Tween 80
that corresponds to the stoichiometric ratio of 10:1
and 9:1, respectively, as well as an amount that
corresponds to one-half of stoichiometric ratio
Y
obs

YN YI expDGH
2
O; N !I mN !ID=RT YU expDGH
2
O;
N !I mN !ID=RT expDGH
2
O; I !U mI !UD=RT
1 expDGH
2
O; N !I mN !ID=RT expDGH
2
O; N !I mN
!ID=RT expDGH
2
O; I !U mI !UD=RT 4
EFFECTS OF NONIONIC SURFACTANTS 1371
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
(5:1) as determined by ITC and uorescence
titration. A set of unagitated samples were also
prepared and served as controls for surface-
induced aggregation. When the amount of Tween
20 added corresponded to its stoichiometric bind-
ing ratio with Albutropin, the concentration of
Tween 20 was approximately 30 mM. This con-
centration was more than three times lower than
the operative CMC for Tween 20 (100 mM) deter-
mined by surface tensiometry. For Tween 80, the
operative CMC was also found to be 100 mM.
Samples were oriented horizontally and agitated
at 600 RPM on a Labnet Orbit 300 orbital shaker
(Woodbridge, NJ) at room temperature (25 28C)
for 96 h. Sample tubes were removed for testing at
0, 24, 48, 72, and 96 h. In addition, nonagitated
control samples were also assayed at each time
point. Prior to testing by SE-HPLC, vials were
centrifuged at 13000 g for 15 min using a refri-
gerated (48C) Savant centrifuge (Holbrook, NY),
and the level of native monomer and the forma-
tion of soluble aggregates were determined using
SE-HPLC.
RESULTS AND DISCUSSION
Fluorescence Quenching
Fluorescence spectroscopy often has been used to
the study of various proteinligand interactions.
It is a sensitive technique that is used to monitor
changes in protein tertiary structure upon ligand
binding and may complement ITC, given its
high sensitivity. The intrinsic uorescence of
the aromatic residues in a protein, particularly
tryptophan and tyrosine, is very sensitive to
their microenvironment. Because ligand binding
usually results in a perturbation of this micro-
environment, the uorescence may be quenched,
providing an effective method to monitor ligand
binding. There are several mechanisms by which
a ligand may induce changes in a proteins intrin-
sic uorescence intensity. First, ligand binding
may cause a protein to undergo conformational
changes that results in loss of resonance energy
transfer between uorophores and, thus, de-
creased uorescence intensity. Second, a ligand
may directly interact or collide with the uor-
ophore and absorb its intrinsic uorescence after
excitation. This process is termed collisional
quenching. Moreover, a ligand may complex with
the protein to form a nonuorescent dark
complex. Such a phenomenon is called static
quenching. Importantly, the magnitude of this
uorescence quenching process has been found to
correlate with the binding afnity between a
protein and its ligand.
20
A number of papers have been published
recently that utilized the intrinsic uorescence of
the single tryptophan residue in HSA as a way to
monitor its binding afnity for fatty acid ligands
and pharmaceutical compounds. For example,
Johansson et al.
23
successfully used uorescence
quenching to investigate the nature of interaction
between HSA and the volatile anesthetic, chloro-
form. Gelamo and Tabak
24
in 2000 also employed
uorescence quenching to arrive at association
constants between BSA, HSA, and a number of
ionic surfactants.
The binding to Albutropinby bothTween20and
Tween 80 was measured by excitation of the two
tryptophan residues in the protein at 295 nm.
There is a shift in the wavelength of maximum
emission (l
max
) from 342 to 340 nm when a
surfactant is added (data not shown), which
indicates that the tryptophan residues are in a
relatively more hydrophobic environment after
binding to the surfactant. Increasing concentra-
tion of surfactant did not result in further shift of
l
max
. In all the experiments, the addition of Tween
to the protein solution decreased the intensity of
the intrinsic tryptophan uorescence. The extent
of uorescence quenching is different for hGH,
HSA, and Albutropin, but all reach a plateau at
relatively low surfactant concentrations. Past
studies have found that a plateau in the ligand-
induced spectrouorometric changes, such as
surfactant induced quenching of tryptophan
intrinsic uorescence, are highly correlated with
saturation of available binding sites on the
protein.
25,26
It appears that the break point in
the uorescence-quenching curve occurs at the
approximate stoichiometry between ligand and
protein.
2326
Interestingly, the steady-state uor-
escence quenching data appear to support the
existence of two saturation points on the binding
isotherm (Figure 1A,B). The rst inection
points observed in the uorescence quenching
isotherms between Tween 20 and Tween 80 with
Albutropin are at approximately 2:1 ratio (Tween
20:Albutropin) and 1:1 ratio (Tween 80:Albu-
tropin), respectively, which corresponds to the
saturation point for the high afnity binding site
(Figure 1A,B). Tween appears to bind to lower
afnity sites on Albutropin after occupying the
stronger binding sites. This observation corrobo-
rates with recent ndings by other researchers
1372 CHOU ET AL.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
that multi-domain proteins possess specic and
distinctive binding sites for small ligands with
different afnities.
2729
The binding isotherms
between the two Tweens and each of the two
domains of Albutropin and the intact fusion
protein are shown on Table 1. Our data show that
the Tween20-proteinbindingstoichiometryis 11:1
(Tween 20:HSA) for HSA, 9:1 (Tween 20:hGH) for
hGH, and 10:1 (Tween 20:Albutropin) for Albu-
tropin. The binding stoichiometry between Tween
80 and Albutropin was found to be 9:1 (Tween T
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Molar Ratio [Tween 20:Albutropin]
0 2 4 6 8 10 12
R
e
l
a
t
i
v
e

F
l
u
o
r
e
s
c
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2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8

Molar Ratio [Tween 80:Albutropin]
R
e
l
a
t
i
v
e

F
l
u
o
r
e
s
c
e
n
c
e
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
0 2 4 6 8 10 12
A
B
Figure 1. (A) Fluorescence quenching isotherm
showing progressive reduction of Albutropin trptophan
intrinsic uorescence intensity with increasing concen-
trations of Tween 20. Error bars represent standard
error of the mean for duplicate samples. Where error
bars are not apparent, the bars were smaller than the
symbol. (B) Fluorescence quenching isotherm showing
progressive reduction of Albutropin trptophan intrinsic
uorescence intensity with increasing concentrations of
Tween 80. Error bars represent standard error of the
mean for duplicate samples. Where error bars are not
apparent, the bars were smaller than the symbol.
EFFECTS OF NONIONIC SURFACTANTS 1373
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
80:Albutropin). The fact that the stoichiometric
binding ratio between Albutropin and Tween does
not correspond to the sum of its two domains is
probably due to changes in the number and/or
conguration of available binding sites upon the
formation of the chimeric protein. Stoichiometric
values derived from a recent crystallographic
analysis found in the literature show excellent
agreement withdata obtained inthe present study
(Table 1); Bhattacharya et al.
30
found a total of
11 binding sites on HSAfor fatty acids with 1014
carbon aliphatic tails (note that the lauric acid
moiety found in Tween 20 has a 12 carbon
hydrophobic group) and up to 7 sites for fatty acids
with longer hydrocarbon chains (Tween 80 con-
tains a 18 carbon hydrophobic tail).
In order to compare the binding afnities for
Tween for each protein, it is necessary to calculate
the dependence of F
0
/F (F
0
and F are the uor-
escence intensities in the absence and presence of
surfactant, respectively) as a function of Tween
concentration. The SternVolmer equation char-
acterizes the interaction in terms of a quenching
constant derived from the slope of the Stern
Volmer plot.
F
0
=F 1 K
SV
Q
F
0
and F are the uorescence intensities before
and after addition of quencher; Qis the concentra-
tion of quencher; K
sv
is the effective quenching
constant of the binding reaction and it is a product
of rate constant for quenching k
q
andlifetime of the
uorophore t
0
.
20
The SternVolmer quenching constants (K
sv
)
obtained for HSA: Tween binding resemble values
published in the literature for HSA and other
surfactants. For example, for HSA binding with
hexadecyl-N,N-dimethyl-3-ammonium-1-propan-
esulfonate (HPS) and cationic cethyltrimethylam-
monium chloride (CTAC) the K
sv
were found to be
(3.53.6) 10
3
and (2.93.1) 10
3
M
1
, respec-
tively.
24
By comparison, the K
sv
for Tween 20 and
HSAwas determinedto be (1.71.8) 10
5
M
1
. K
sv
has a strong correlation with binding afnity
between the quencher (surfactant in this case)
and a given protein. In fact, when the quenching
is completely static in nature due to formation of
nonuorescent complex, K
sv
is the afnity con-
stant.
22
Until this work, there has been no attempt to
measure the binding of surfactants to hGH using
uorescence quenching. Table 1 lists the Stern
Volmer quenching constant obtained for Albutro-
pin and each of its two domain proteins. It appears
that Tween 20 has a much higher afnity for HSA
than hGH. The difference in association constants
(K
sv
) between HSA and hGH is approximately two
orders of magnitude. For Albutropin, the Stern
Volmer quenching constant of the high-afnity
site was (2.02.1) 10
5
for Tween 20 and (1.8
2.0) 10
5
M
1
for Tween 80.
ITC Binding Studies
Although its sensitivity is less than that of uor-
escence titration, ITC provides direct thermo-
dynamic information about ligand binding to
protein.
31
ITC measures the heat associated with
binding of a ligand to macromolecule. In a single
experiment, the values of binding constant (K
a
),
the stoichiometry (n), the enthalpy of binding
(DH
b
), and the entropy of binding (DS) can be
determined.
31,32
ITC experiments with hGH, HSA, and Albutro-
pin were performed to determine whether binding
parameters obtained by this method agree with
those obtained from uorescence titrations. The
association parameters derived from uorescence
titration differs from those determined by ITC
(Table 1). Since two completely different phenom-
ena are being measured, it is not surprising that
there are differences in the calculated binding
afnity. However, it is important to note that the
rankordering of the binding afnity is the same for
both methods, with Albutropin having the highest
binding afnity for Tween 20, followed by HSA,
then hGH. Due to the weak interaction between
Tween 20 and hGH, it is not possible to determine
the bindingconstant usingITC. Althoughprevious
studies have shown that nonionic surfactants
binds with hGH via hydrophobic interaction,
Tween appears to exhibit higher afnity for the
HSA domain of Albutropin as evidenced by the
association constants derived from these experi-
ments. A comparison of the ITC binding isotherm
observed in this study with hGH shows that our
result matches previously published data by Bam
et al.
6
In fact, the binding stoichiometry between
hGH and Tween 20 we found is identical to the
value obtained by Bam et al.
5
via refractive index
studies.
Importantly, ITC yields the same protein-
surfactant stoichiometry as the uorescence titra-
tion experiments (Table 1). Analysis of the binding
isotherm by established models for each method
indicate that there are specic sites to which
surfactants bind that can be saturated (Figures 1
and 2B). This is demonstrated by both the plateau
1374 CHOU ET AL.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
in the isothermgenerated by uorescence quench-
ing as well as the progressively smaller enthalpic
signal from ITC titration (Figures 1 and 2A).
Moreover, it was found that ITC and uorescence
titration data were best t using a binding site
model with two sites of different afnities. As one
can see in Table 1, for Tween 20, the association
constant for the high afnity and the low afnity
site of Albutropin as determined by ITC is (2.1
2.2) 10
4
and (1.11.2) 10
3
M
1
, respectively.
Essentially, the high afnity site of Albutropin is
bound by Tween 20 over 20 times more avidly than
the lowafnity site. For Tween 80, the association
constant is (1.41.5) 10
4
M
1
for the highafnity
site and (1.21.3) 10
3
M
1
for the low afnity
site. As early as the 1970s, researchers have
recognized the ability of HSA to assume different
transient solution conformations upon fatty acid
binding. Given its natural biological role, HSA is
known to exhibit much exibility in solution and
this property is believed to be responsible for its
ability to bind a diverse array of ligands.
16
Spector
termed this property of HSA congurational
adaptability and it was found that fatty acids
with 12 carbon hydrophobic tails exhibit the
highest afnity for HSA.
17
Since the hydrophobic
tail of Tween 20 contains 12 carbons versus 18
in Tween 80, Tween 20 probably has a greater
number of productive interactions with binding
pockets onAlbutropin. The overall lower afnity of
Tween 80 for Albutropin may be due to the longer
hydrophobic tail of Tween 80, which may cause
steric hindrance at the high afnity binding site.
Agitation Study
Agitation is a frequently encountered source of
stress for protein pharmaceuticals, especially for
liquid formulations. During vial lling, shipment,
reconstitution, and administration, agitation of
the solution could repeatedly expose the protein to
denaturing surfaces, such as air-liquid and solid-
liquid interface. If a sub-CMC concentration of
Tween is sufcient to prevent agitation-induced
aggregation under the strongly destabilizing con-
ditions employed in this study, it is a good indi-
cator that a protein-specic, rational approach to
surfactant formulation could be accomplished.
The ability of Tween to protect Albutropin
against agitation-induced damage is an important
formulation consideration. If it does protect, is this
effect due to specic binding to the protein or by
saturating the air-water interface or bothmechan-
isms? The stability of Albutropin during agitation
was monitored using size-exclusion chromatogra-
phy (SE-HPLC). After Albutropin (at pH 7) was
agitated for 24 h, there was detectable conversion
of the monomeric protein into soluble aggregates
(Figure 3). The loss of monomer occurs concur-
rently with an increase in the amount of dimer,
trimer, and large oligomer (that elutes with the
void volume). After 96 h, the level of monomeric
protein decreased from99%to 30%(Figure 5A). In
samples withTween 20: Albutropinmolar ratios of
5:1, the loss of monomer is delayed relative to the
0 10 20 30 40 50 60 70 80
-10
-8
-6
-4
-2
Time (min)

c
a
l
/
s
e
c
0 5 10 15 20 25
- 1.2
- 1.0
- 0.8
- 0.6
- 0.4
- 0.2
0.0
Molar Ratio
k
c
a
l
/
m
o
l
e

o
f

i
n
j
e
c
t
a
n
t
A
B
Figure 2. (A) Raw isothermal titration calorimetry
(ITC) data for Albutropin titration with Tween 80.
(B) ITC isotherm for the binding of Tween 80 to
Albutropin. After integrationof the rawsignal, enthalpy
of binding of each injection was obtained and plotted
versus molar ratio of Tween 80: Albutropin.
EFFECTS OF NONIONIC SURFACTANTS 1375
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
samples with no Tween. However, aggregation
still proceeds to a signicant extent by the end of
the study. By contrast, at a stoichiometric ratio
of 10:1 Tween 20: Albutropin, aggregation was
essentially completely inhibited, even after 96 h
of continuous agitation (Figure 5A). In order to
ensure that aggregation was not simply due to
storage of samples at 258C, nonagitated controls
without Tween 20 were stored at 258C for the
duration of the agitation study. After 96 h of
storage at the aforementioned temperature, there
was no loss of monomeric Albutropin in the control
samples.
It is important to note that surfactant CMC is
highly dependent on solution conditions, such as
ionic strength, temperature, pH, and other solu-
tion components such as excipients, as well as
the presence of protein.
1
The implication of this
ndingis that evenwhenaCMClevel of surfactant
is desired, the true CMC of the protein solution
may not be known unless effort is undertaken to
detect the existence of surfactant-protein interac-
tion and its effect on the operative CMC. When a
decision must be made regarding how much sur-
factant is to be added to a formulation, it may be
hazardous to utilize the published CMCvalues for
a particular surfactant, as the CMC value may be
underestimated in these cases. To ensure that,
under the conditions of this study, the concen-
tration of surfactant is below the operative CMC
when added at a stoichiometric ratio with Al-
butropin, surface tensiometry was employed to
measure the effective CMC with the protein
present in solution. Bam et al.
5
using electron
paramagnetic resonance (EPR) found that appar-
ent surfactant CMC in the presence of hGH
was shifted to lower Tween concentrations. This
phenomenon was attributed to the formation of
mixed-surfactant protein aggregates at a surfac-
tant concentration below the reported CMC. The
reported CMC values of Tween 20 and Tween 80
are 59 and 12 mM, respectively, in pure water at
258C.
33
In the present study, the apparent CMCof
Tween 20 and Tween 80 were both shifted to over
100 mM (data not shown). It is possible that in the
presence of Albutropin, the concentration of
Tween at the air/water interface decreases as the
surfactant is bound onto the protein and drawn
into the bulk solution; as a result, the amount of
surfactant needed to reach CMC is increased.
Barreiro-Iglesias et al.
34
recently observed the
same phenomenon when they studied the interac-
tion between Tween 80 and carbopol
1
polymers.
The authors attributed the increase in CMC in
their systemto direct polymer-surfactant binding.
This nding further supports our hypothesis that
Tween binds Albutropin. At the concentration of
protein used (0.3 mg/mL) a 10:1 molar ratio of
Tween 20 corresponds to a concentration of about
30 mM, which is about one-third of the effective
CMC measured by surface tensiometry. Increas-
ing the concentration of Tween 20 to >30:1 molar
ratio (above the CMC) confers the same degree of
protection against agitation-induced aggregation
as at 10:1. This demonstrates that a stoichiometric
amount of Tween 20 is sufcient to protect Al-
butropin against agitation-induced aggregation.
Similar results were obtained using Tween 80
as the surfactant (Figure 4). As can be seen
on Figure 5B, both samples that contained Tween
80 at 9:1 molar ratio (surfactant: protein) and
samples with Tween 80 just above CMC did not
form soluble aggregates throughout the studys
duration.
One potential advantage of minimizing the
amount of surfactant used is that it is well known
that Tween surfactants can contain a low level of
residual peroxide, which can potentially affect the
stability of oxidation-sensitive proteins. Surfac-
tants are also known to be susceptible to degrada-
tion during storage; and the resulting formation
of oxidizing free radicals may compromise both
physical and chemical stability of the protein



0 hours
24 hours
48 hours
72 hours
4 5 6 7 8 9 10 11


6 7 8 9

A
b
s
o
r
b
a
n
c
e

(
2
8
0

n
m
)

Elution time (minutes)
soluble
aggregates
monomer
fragment
Figure 3. Size-exclusion chromatography (SEC) data
indicating time-dependent loss of monomer and forma-
tion of soluble aggregates during agitation in the
absence of Tween.
1376 CHOU ET AL.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
during long term storage.
35
The results of the
current study demonstrate that it is not always
necessary to obtain a complete coverage of the
air-water interface with above-CMC concentra-
tion of surfactants to maximally inhibit agitation-
induced aggregation, if direct binding of the sur-
factant to the proteinoccurs. Binding of surfactant
can inhibit aggregation either through a reduction
in intermolecular contact between protein mole-
cules and/or increased protein conformational
stability.
Equilibrium Unfolding Study
To gain further insight into the mechanism for
inhibition of agitation-induced aggregation by
Tweens, the free energy of unfolding was mea-
sured in the absence and presence of surfactant.
Since the rst step in surface-mediated protein
aggregation is the adsorption and unfolding of the
protein at the interface, we hypothesize that one
of the mechanisms by which a sub-CMC level of
Tween stabilizes Albutropin against agitation in-
duced aggregation is by increasing the free energy
of protein unfolding. The ability of Tweens to
retard aggregation may be due to thermodynamic
stabilization of the native state, thereby decreas-
ing the population of partially unfolded, aggrega-
tion-competent species in solution.
36,37
As can be
seen in Figures 6 and 7, the addition of Tween 20
and Tween 80 at stoichiometric ratio (10:1) and
(9:1), respectively, resulted in a shift in unfold-
ing curves to higher GdnHCl concentrations.
These results suggest that binding of Tween to
the native state of Albutropin increases the free
energy of unfolding.
The unfolding curve displays two distinct un-
folding events, presumably due to unfolding of the
Time (hours)
0 20 40 60 80 100 120
P
e
r
c
e
n
t

R
e
c
o
v
e
r
y

o
f

S
o
l
u
b
l
e

M
o
n
o
m
e
r
0
20
40
60
80
100
Time (hours)
0 20 40 60 80 100 120
P
e
r
c
e
n
t

R
e
c
o
v
e
r
y

o
f

S
o
l
u
b
l
e

M
o
n
o
m
e
r
0
20
40
60
80
100
A
B
Figure 5. (A) Percent recovery of soluble monomeric
protein as a function of duration of agitation. Filled
circle, control (no Tween); open circle, 5:1 molar ratio
[Tween 20:Albutropin]; lled triangle, 10:1 molar ratio
[Tween 20:Albutropin]; open triangle, Tween 20 at cri-
tical micelle concentrations (CMC). Each point repre-
sents the mean of three vials standard deviation.
Where error bars are not apparent, the bars were
smaller than the symbol. (B) Percent recovery of soluble
monomeric proteinas a functionof durationof agitation.
Filled circle, control (no Tween); open circle, 9:1 molar
ratio [Tween 80:Albutropin]; lled triangle, Tween 80 at
CMC. Each point represents the mean of three vials
standard deviation. Where error bars are not apparent,
the bars were smaller than the symbol.
Figure 4. SEC data indicating no time-dependent
loss of monomer and formation of soluble aggregates
duringagitationinthe presence of Tween80at 9:1molar
ratio [Tween 80:Albutropin].
EFFECTS OF NONIONIC SURFACTANTS 1377
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
two individual domains (the HSA and hGH por-
tions). Thermodynamic analysis of these curves
requires that each transition behaves as a two-
state model, whereby the unfolding is reversible.
In order to demonstrate the reversibility of the
unfolding process, far UV CD scans were taken
after exhaustive dialysis against buffer was per-
formed to remove GdnHCl from samples that
contained 6Mof the denaturant. It was found that
unfolding of Albutropinwas >93%reversible (data
not shown). In their 1996 paper Bam et al.,
7
using
electron paramagnetic resonance (EPR) spectro-
scopy found that Tween could stabilize and popu-
late a partially unfolded intermediate of hGH at
4.5M guanidine hydrochloride. In the current
study, such a folding intermediate of the hGH
domain was not observed with the addition of
Tween. This discrepancy is most likely due to the
fact that the chosen spectroscopic method (Far UV
CD spectroscopy) is primarily used to monitor
changes inproteinsecondary structure. Since EPR
spectroscopy is used to monitor changes in both
secondary and tertiary structure of a protein, it
may explain why such a stable intermediate was
observed in the earlier study.
7
It should be noted
that the most signicant spectroscopic transitions
found by Bam et al. occurs over a narrow range of
guanidine concentrations, just as we have ob-
served with Albutropin.
A comparison of the denaturation midpoint as
well as the free energy of unfolding values withthe
literature suggests that the rst part of the curve
corresponds to the unfolding of the HSA domain,
while the second transition event agrees with
the values derived from unfolding of the hGH
domain.
7,38,39
Observation of multiple-unfolding
events has been observed for proteins with sepa-
rate domains. Since the unfolding curves of
Albutropin do not follow the traditional single
two-state model and appears to have a signicant
plateau between two separate unfolding events,
thermodynamic parameters could not be obtained
by the linear extrapolationapproachoftenused for
protein that have a two-state unfolding behavior.
Analysis of unfolding curves with multiple transi-
tions has been a great challenge until recently. In
2001, Hung and Chang reported a formal deriva-
tion of a mathematical model that describes the
folding-unfolding process involving multiple inter-
mediates (Equation 4).
21
The model was developed
to analyze the unfolding data for human placental
alkaline phosphatase and could be applied to any
other multi-state unfolding data. The Hung and
Chang model takes into account the equilibrium
[Guanidine HCI] (M)
0 2 4 6
F
a
r

U
V

C
D

S
i
g
n
a
l

a
t

2
2
2

n
m

-20
-15
-10
-5
0
Figure 6. Unfolding curves for Albutropin at 0.1 mg/
mL in the absence and presence of Tween 20. Unfolding
curves were measured using Far UV CD spectroscopy
monitoring the loss of protein helical structure at
222 nm. Open circle, control (no Tween 20); lled circle,
Tween 20 10:1 molar ratio [Tween 20: Albutropin]. Each
point represents the mean of three samples standard
deviation. Where error bars are not apparent, the bars
were smaller than the symbol.
[Guanidine HCI] (M)

0 2 4 6
F
a
r

U
V

C
D

S
i
g
n
a
l

a
t

2
2
2

n
m


-20
-15
-10
-5
0
Figure 7. Unfolding curves for Albutropin at 0.1 mg/
mL in the absence and presence of Tween 80. Unfolding
curves were measured using Far UV CD spectroscopy
monitoring the loss of protein helical structure at
222 nm. Open circle, control (no Tween 80); lled circle,
Tween 80 9:1 molar ratio [Tween 80: Albutropin]. Each
point represents the mean of three samples standard
deviation. Where error bars are not apparent, the bars
were smaller than the symbol.
1378 CHOU ET AL.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
constants between the various folding intermedi-
ates and bases its assumptions on the linear free
energy changes theory developed by Santoro and
Bolen.
20,40
Using this model, our analysis shows that the
denaturation midpoint (D
0.5
) in GdnHCl solutions
of the rst unfolding event was increased from
1.8 0.1 M GdnHCl in the absence of Tween 20 to
2.1 0.2 M in the presence of Tween 20 (Table 2).
It was determined that the addition of stoichio-
metric amount of Tween 20 results in an increase
in thermodynamic conformational stability of the
HSA domain of Albutropin by 1.3 0.5 kcal/mol.
By comparison, a stoichiometric amount of Tween
80 conferred about 0.6 0.2 kcal/mol of stabiliza-
tion to Albutropin (Table 2). In both cases, the free
energy of unfolding of the second unfolding event,
presumably involving the hGH domain, was not
signicantly altered. This observation may be due
to the fact that both Tween 20 and Tween 80
appear to bind more strongly to the HSAdomain of
Albutropin, thus reduce the effect of these surfac-
tants have on stability of the hGH domain of the
fusion molecule.
These results, along with the Tween binding
studies, indicate that Tween stabilizes Albutropin
against agitation-induced aggregation by prefer-
entially binding to the native state of the protein,
mostly to the HSA domain. This effect can be best
explained by the Wyman linkage function, which
states that differential binding afnity of a ligand
in a two-state equilibrium will shift the equili-
briuminfavor of the state withthe greater binding
afnity.
37,41,42
Spectroscopic studies of Albutropin
both in the presence and absence of Tween 20 and
Tween 80 in buffer showed complete retention of
native protein secondary and tertiary structure
(data not shown). Thus, Tween stabilizes Al-
butropin by preferentially binding to the native
molecule, which results in greater protein con-
formational stability; this means that as a popula-
tion, fewer Albutropin molecules are in expanded,
aggregation-competent conformations that can
aggregate upon exposure to agitation.
Previous reports have shown that Tween could
protect protein against aggregation via several
mechanisms simultaneously. In addition to stabi-
lizing the native state of Albutropinandcompeting
for occupation of the air-water interface, Tween 20
Tween 80 may prevent protein aggregation by
binding to hydrophobic sites on the proteins
surface. The result of this interaction is that less
aggregation prone-hydrophobic areas on the pro-
tein are exposed to the solvent and thus the
probability of proteinprotein interaction is
decreased. Tween could inhibit intermolecular
contact by occupying the hydrophobic regions of
the protein with its nonpolar aliphatic tail. The
driving force for this type of interaction is the
hydrophobic effect.
6
The importance of this mech-
anismwas illustrated withhGHwhenit was found
that despite its weak binding to Tween, the opti-
mal level of protection against agitation-induced
aggregation was rendered when the protein was
formulated with sufcient surfactant to saturate
all the binding sites on the proteins surface.
CONCLUSIONS
We have evaluated the utility of nonionic surfac-
tants Tween 20 and Tween 80 as excipients for
prevention of surface-induced denaturation and
aggregation of recombinant fusion protein, Al-
butropin. The results of this work show that
Tween 20 and Tween 80 both confer signicant
Table 2. Thermodynamic Parameters Obtained from Guanidine Hydrochloride
Unfolding Studies
Surfactant Used
Thermodynamic
Parameters
(GdnHcl)
D
0.5
M
DG of Unfolding
Kcal/mol
DDG
Kcal/mol
With Tween 20 Transition 1 2.10.2 5.30.7 1.30.5
Transition 2 4.40.3 5.80.8 N/S
No Tween 20 Transition 1 1.80.1 4.00.2
Transition 2 4.50.2 5.90.6
With Tween 80 Transition 1 2.00.1 4.80.4 0.60.2
Transition 2 4.40.3 5.90.5 N/S
No Tween 80 Transition 1 1.90.2 4.30.3
Transition 2 4.40.2 5.90.5
Data presented are the means and standard deviations obtained from triplicate measurements.
N/S, not signicant.
EFFECTS OF NONIONIC SURFACTANTS 1379
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 94, NO. 6, JUNE 2005
protection against agitation-induced damage to
Albutropin, even at concentrations below the
CMC, provided the stoichiometric molar ratio
identied by the binding studies is used. Tween
protects Albutropin against aggregation by
increasing thermodynamic stability of the pro-
tein, thus decreasing its propensity for denatura-
tion and subsequent aggregation. Despite the
relatively weak interaction, binding of Tween
20 and Tween 80 by Albutropin provides 0.6
1.3 kcal/mol of stabilization to the protein. Sa-
turation of the hydrophobic surface areas of
Albutropin by Tween may also provide steric
hindrance by decreasing intermolecular contacts
that precede the onset of aggregation. Finally, the
current study provides strong evidence that a
rational approach to surfactant formulation of
protein pharmaceuticals could minimize the risks
associated with a given excipient, yet provide
sufcient benet on protein stability.
ACKNOWLEDGMENTS
We greatly appreciate the generous gift of Al-
butropin from Human Genome Sciences, Inc. This
work was partly supported by HGSI and NIH
Leadership in Biotechnology Fellowship given to
DKC.
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