Advanced Drug Delivery Reviews 63 (2011) 1160–1171

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Advanced Drug Delivery Reviews

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a d d r

Molecular origins of surfactant-mediated stabilization of protein drugs☆

Hyo Jin Lee a, b, Arnold McAuley b, Karl F. Schilke a, Joseph McGuire a,⁎
a
School of Chemical, Biological and Environmental Engineering, Oregon State University, Corvallis, OR 97331
b
Process and Product Development Department Amgen Inc., Thousand Oaks, CA 91320

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

Article history: Loss of activity through aggregation and surface-induced denaturation is a significant problem in the
Received 30 March 2011 production, formulation and administration of therapeutic proteins. Surfactants are commonly used in
Accepted 29 June 2011 upstream and downstream processing and drug formulation. However, the effectiveness of a surfactant
Available online 6 July 2011
strongly depends on its mechanism(s) of action and properties of the protein and interfaces. Surfactants can
modulate adsorption loss and aggregation by coating interfaces and/or participating in protein-surfactant
Keywords:
Adsorption
associations. Minimizing protein loss from colloidal and interfacial interaction requires a fundamental
Aggregation understanding of the molecular factors underlying surfactant effectiveness and mechanism. These concepts
Formulation provide direction for improvements in the manufacture and finishing of therapeutic proteins. We summarize
Protein the roles of surfactants, proteins, and surfactant-protein complexes in modulating interfacial behavior and
Stabilization aggregation. These events depend on surfactant properties that may be quantified using a thermodynamic
Surfactant model, to provide physical/chemical direction for surfactant selection or design, and to effectively reduce
aggregation and adsorption loss.
© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161
2. Managing protein aggregation and adsorption loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161
2.1. Mechanisms of aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161
2.1.1. Concentration-induced aggregation (Mechanism 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1161
2.1.2. Aggregation induced by conformational change (Mechanism 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162
2.1.3. Aggregation induced by chemical reaction (Mechanism 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1162
2.1.4. Nucleation-dependent aggregation (Mechanism 4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163
2.1.5. Surface-induced aggregation (Mechanism 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163
2.2. Surfactants used in drug products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164
2.2.1. Polysorbates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164
2.2.2. Poloxamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1164
2.3. Surfactant modulation of protein adsorption and aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165
2.3.1. Protein stabilization by surfactant adsorption at interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165
2.4. Protein stabilization by surfactant-protein association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1167
2.4.1. A simple view of surfactant-protein mixtures at the air-water interface. . . . . . . . . . . . . . . . . . . . . . . . . . 1167
3. A testable, thermodynamic argument to guide surfactant selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1167
3.1. Insights gained from intact and protein-depleted pulmonary surfactant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1167
3.2. Surfactant-protein association at surfactant concentrations above the CMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1168
3.3. Surfactant-protein association at surfactant concentrations below the CMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1168
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1169
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1169
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1169

☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Formulating Biomolecules: Mechanistics Insights in Molecular Interactions”.
⁎ Corresponding author at: School of Chemical, Biological and Environmental Engineering, Oregon State University, 103 Gleeson Hall, Corvallis, OR 97331–2702. Tel.: + 1 541 737
6306; fax: + 1 541 737 4600.
E-mail address: mcguirej@engr.orst.edu (J. McGuire).

0169-409X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.addr.2011.06.015
 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171
1. Introduction
In recent years, the number of protein and peptide therapeutics
reaching the marketplace has increased significantly for most major
pharmaceutical and biotechnology companies. Technology has ad-
vanced greatly since the development of recombinant human insulin,
the first medicine to be commercially produced by DNA cloning and
manipulation [1]. Since then, rapid developments in biotechnology
have enabled the commercial production of various hormones, blood
factors, cytokines, and fully human monoclonal antibodies. Such
therapeutic proteins are widely used to manage and treat hemophilia,
cancer, diabetes, hepatitis, inflammation and other ailments. Thera-
peutic proteins are typically based on complex polypeptides, large
molecules that are made up of a well-defined sequence of amino acids,
and may be produced through a combination of chemical or biological
means. Proteins adopt distinct three-dimensional structures that are
usually necessary for correct function, but which are often highly
sensitive to the surrounding environment and may be easily distorted
or altered. Although protein drugs are generally considered to have
fewer inherent side effects than traditional chemical agents, they are
usually very surface-active and are more susceptible to activity loss
through adsorption, structural unfolding and aggregation than small
molecules. This is a substantial problem for the biopharmaceutical
industry, because losses of biological activity through aggregation or
surface-induced structural alteration (denaturation) are encountered
throughout the production, formulation and administration of
therapeutic proteins. Therefore, considerable efforts have been
made to identify the causes of adsorption loss and aggregation, and
to develop effective methods to minimize these detrimental effects
and associated costs. Several mitigation strategies are used in the
biotechnology industries to stabilize therapeutic proteins, but the
addition of surfactants appears to be a general approach [2].
Proteins can often be stabilized against adsorption loss through the
use of properly-chosen surfactants. Preferential location of surfactant
molecules at interfaces, such as the walls of glass vials or the surfaces
of bubbles, may strongly inhibit adsorption of proteins and prevent
their subsequent denaturation or loss. In addition, formation of
surfactant-protein complexes in solution can reduce the surface
activity of the proteins, thus stabilizing them toward close approach
and aggregation. Aggregation can also be inhibited by a variety of non-
surfactant stabilizers, which are selected based on their ability to
inhibit specific molecular mechanism(s) that govern aggregation
phenomena in a given system. In Section 2, below, we briefly outline
the major mechanisms that contribute to aggregation and surface-
induced conformational changes. Some chemical strategies used to
inhibit or slow those mechanisms, including the use of surfactants, are
also presented. The stabilization of proteins by surfactants, in the
presence of interfaces, is discussed in Section 3. Particular emphasis is
given to the mechanisms by which complexes of protein and surfactant
molecules might influence thermodynamic barriers, leading to stabili-
zation of the proteins against aggregation and adsorption loss.
2. Managing protein aggregation and adsorption loss
Aggregation phenomena in protein therapeutics have been studied
and reported extensively by academia, industry and regulatory
agencies. Aggregation is highly undesirable due to the profound
impact on the stability of the drug product, which can result in loss of
activity, unwanted immunogenic responses, and increased rate of
rejection as a marketable product [3]. Several workers have reported
on the different mechanisms of aggregation, and suggest possible
methods to inhibit aggregation [4–6]. Various external chemical or
physical factors may be responsible. Additionally, the inherent
properties of the protein (e.g. charged or hydrophobic regions) may
make it unusually susceptible to aggregation. In such cases, ag-
gregation can often be inhibited by modifying the molecular properties
1161
or by changing the external environment [7,8]. Inherent properties can
be effectively modified by site-directed mutagenesis [7] or chemical
modification (e.g. PEGylation) [8], but such modifications may
compromise the biological activity of the protein. Thus, the simplest
and most common method of inhibiting aggregation is to change the
nature of the environment surrounding the protein by adjusting
solution conditions such as pH, or by adding stabilizers/excipients. By
carefully examining the mechanism(s) responsible for the aggregation,
we can identify specific changes or stabilizing molecules that will
effectively inhibit that mechanism via a molecular-level interaction,
and thus enhance the stability of the formulation.
2.1. Mechanisms of aggregation
Protein aggregation occurs under different stress conditions, and
produces unwanted and detrimental effects on a therapeutic drug
product. Aggregation occurs through several different major mecha-
nisms and pathways (Fig. 1), discussed in detail in the examples
below. Although a particular mechanism may identify an aggregation
pathway for a particular protein, it may not be relevant for another
protein. Also, more than one mechanism or pathway may be simul-
taneously responsible for destabilizing a protein formulation [5]. A
fundamental understanding of the mechanisms of aggregation is
not only valuable in identifying the cause of a problem, but also helpful
in suggesting methods to suppress aggregation to an acceptable level.
It may be noted that aggregation may or may not lead to precipitation
or insoluble aggregates.
2.1.1. Concentration-induced aggregation (Mechanism 1)
Because proteins tend to be surface-active due to their polymeric
structure and amphipathic nature [9], they can form reversible
aggregates especially in high concentration formulations. Mechanism
1 begins with an association of native monomers into an initially-
reversible complex. As protein concentration increases or time passes,
the protein complex may become an irreversible aggregate (Fig. 1,
scheme 1). Formation of intermolecular disulfide linkages (possibly
through disulfide interchange reactions) is one cause of this irrevers-
ibility [5]. IgG antibodies have been observed to form reversible soluble
aggregates in high concentration solutions. Electrostatic interactions
and hydrogen bonds contribute to the self-association of IgG molecules.
Hydrophobic patches in the Fc region of IgG are considered to be a
major factor in inducing aggregation at higher concentrations [10].
Human interleukin-1 receptor anatagonist (IL-1ra) is part of the
IL-1/Fibroblast Growth Factor family of proteins with a predominantly
β-strand secondary structure. It self-associates and aggregates
without changes to secondary structure at high concentrations and
elevated temperatures. This self-association induced aggregation was
attributed to a positively-charged Lys96 residue in the IL-1ra molecule.
Aggregation affinity was dependent on the buffer ionic strength and
the type of anion. Phosphate anion was found to inhibit aggregation
more weakly than citrate or pyrophosphate at pH 6.5. Proteolytic
removal of an unstructured N-terminal region containing another
lysine residue also substantially reduced the rate of self-association. It
was proposed that the anions compete for cationic sites on the
protein, preventing the formation of cation-π interactions between
protein molecules. Based on pK values at 25 °C, citrate and
pyrophosphate anions would have 2 to 3 times more ionized groups
than phosphate at pH 6.5. The relative affinity of the anion binding to
the cationic site (and hence, inhibition of aggregation) was correlated
with the number of ionizable groups at a given pH [7].
Insulin aggregation is generally considered reversible at room
temperature near its isoelectric point. This can be attributed to
electrostatic interactions due to the marked charge anisotropy of
the polypeptide. Addition of heparin, a highly charged polyanion,
prevented aggregation at pH higher than 6 by binding to the positive
domains of insulin to form heparin-insulin complexes. Heparin was
1162 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171
Fig. 1. Schematic illustration of five common mechanisms of aggregation. Multiple mechanisms may be at work in any given system. Adapted with permission from [5]. Copyright ©
2009 Bentham Science Publishers.
also able to dissociate aggregate particles of insulin, indicating that the
association was largely charge-based [11].
2.1.2. Aggregation induced by conformational change (Mechanism 2)
Another very common form of aggregation occurs when non-native
states of the protein have a higher affinity with each other than the
native state. In Mechanism 2, proteins aggregate after they go through a
conformational change or partial unfolding (denaturation), which is the
rate-limiting step (Fig. 1, scheme 2). Interactions between the
denatured proteins are typically driven by hydrophobic associations,
and are usually strong enough to be practically irreversible. External
factors like heat and shear can induce the protein into a non-native state,
as is commonly observed with the proteins in egg whites.
Stability of interferon-tau (INF-tau), which is a novel type 1
interferon, depended on the type of buffering agent, even when the
pH and ionic strength were kept constant. At pH 7.0, INF-tau in Tris
and phosphate buffers at elevated temperatures was observed to
aggregate, with a substantial loss of tertiary structure and slightly
expanded non-native conformation. However, samples containing
free histidine as a buffering agent suppressed thermally-induced
aggregation, and little loss of tertiary structure was observed at the
same pH. Detectable binding was observed only for histidine, and only
to the native conformation. Histidine had little effect on protein-
protein repulsion, suggesting that colloidal stabilization was unim-
portant. Thermodynamic stabilization was achieved by binding of
histidine to a specific ligand in the native state of INF-tau and thus,
maintains its native state and suppresses aggregation [12].
While normally a stable drug product, samples of recombinant
human granulocyte colony stimulating factor (rhGCSF) were observed
to aggregate under physiological conditions [13]. Added sucrose was
able to stabilize rhGCSF, and inhibited aggregation under stressed
conditions. The thermodynamic stability of rhGCSF increased with the
addition of sucrose, which is preferentially excluded from the surface
of the protein [14,15]. In this system, sucrose acted as a stabilizer by
shifting the equilibrium to favor the native compact species rather
than the structurally expanded species.
2.1.3. Aggregation induced by chemical reaction (Mechanism 3)
Mechanism 3 is similar to Mechanism 2, but the conformational
change is caused by chemical modification or degradations such as
oxidation, deamidation, or disulfide scrambling (Fig. 1, scheme 3).
Chemical changes may profoundly alter protein properties such as
solvent accessibility of hydrophobic patches, reduction in electrostatic
repulsion due to modification of charged residues, or disruptions of
 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171
the native structure that trigger unfolding. It is important to note that
chemically different species are not necessarily degradation products,
but may be formed during normal production of a drug. Truncated
peptides or under-glycosylated glycoproteins may be more suscep-
tible to aggregation than their correctly-formed counterparts [5].
The stability of a basic leucine zipper (bZIP) domain of activating
transcription factor 5, which consists of a single α-helix and a single
cysteine residue, was observed to be dependent on intermolecular
disulfide bonds which stabilize the native structure. Reduction of the
disulfide bond resulted in the unfolding of the peptide and exposed
hydrophobic regions, which resulted in aggregation of the protein [16].
A detailed structural characterization of the effects of methionine
oxidation on the stability of the human IgG Fc region was studied.
Oxidation of methionine can generate a repulsive interaction between
the side chains of methioninine residues in the CH2 and CH3 domains,
and thus disrupt the native structure. Although Met residues in both
CH2 and CH3 domains were affected by oxidation, more structural
perturbations were observed in the CH2 domain. Therefore, an
increase in aggregation would be likely due to the structural
instability of the CH2 domain of the Fc region. Since aggregation was
observed only for highly oxidized proteins, addition of excipients such
as methionine or sodium thiosulfate that acts as oxygen scavengers
would be sufficient in preventing methionine oxidation that can cause
aggregation [17].
2.1.4. Nucleation-dependent aggregation (Mechanism 4)
In contrast to the three previous mechanisms, which are based on
interactions between individual protein molecules, protein aggrega-
tion can also be attributed to nucleation-dependent processes.
Mechanism 4 describes an aggregation process that is initiated
when a “critical nucleus” is formed in solution, and native proteins
are recruited, and often partially unfolded, to form aggregated species
(Fig. 1, scheme 4). The process is not unlike the growth of a large
crystal from a supersaturated solution after addition of a seed crystal.
In this case, the “seed” is a microscopic aggregated particle of a
denatured or otherwise non-native conformation. A “lag phase”
(often weeks or months) is characteristic of this mechanism. During
this lag phase (which may vary considerably from sample to sample),
the seed nucleus grows, but no particles or precipitation can be
observed. After the formation of a critical nucleus, the aggregation
progresses rapidly, with the relatively sudden formation of visible
aggregates or precipitates in solution. These nuclei may be denatured
proteins, or solid contaminants (e.g. particles of silica from vials or
metal from pumps) [5].
An excellent example of this nucleation-dependent mechanism is
the 10-residue peptide of human amylin, which is used as a model
system to study self-assembly of amyloid fibril proteins. Amylin was
observed to aggregate in response to low levels of asparagine
deamidation, such as might be found as impurities in synthetic
amyloid peptides. Seeding solutions of the native peptide with small
amounts of deamidated peptide resulted in rapid aggregation to form
characteristic amyloid structures. Additionally, when the affected side
chain of the deamidated peptide is deprotonated and negatively
charged (at physiological pH), electrostatic interactions with the
positively-charged N-terminus of another amylin peptide induce the
propagation of the aggregation event. A relevant point is that
phosphate anion is known to promote deamidation of Asp/Gln
residues [18].
Tungsten contamination from a needle tip was observed to induce
significant protein aggregation in pre-filled syringes. Tungsten
microparticles become soluble at lower pH, forming tungsten
polyanions which are able to precipitate a monoclonal antibody
(mAb) within seconds. The tungsten polyanions bind to the proteins,
reducing the net charge and screen the electrostatic repulsions
between the native monomers to induce precipitation. However, at
pH 6.0 and higher, tungsten polyanions do not form and aggregation
1163
was not observed. The authors caution that the small number of
tungsten particles required to induce precipitation of the antibodies,
combined with poor mixing in the needle, precluded defining an
acceptable syringe volume for a given protein [19].
Silicone oil, a common lubricant in pharmaceutical applications,
has also been implicated in aggregation of monoclonal antibodies in
pre-filled syringes. Although silicone oil alone did not induce
aggregation, a synergistic effect producing substantial aggregation
was observed when samples were agitated in the presence of silicone
oil. Perturbation of the monomeric state of the protein by a
combination of air-water and oil-water interfacial stresses was
implicated in the aggregation. Complete inhibition of silicone oil-
induced protein loss was observed when the nonionic surfactant
polysorbate 20 was added (polysorbates are discussed in more detail
in Section 2.2.1, below). Polysorbate 20 is known to compete with
protein molecules at air-water and oil-water interfaces (model
hydrophobic systems), where it prevents structural perturbations
and subsequent aggregation of the protein molecules at the
unprotected interfaces [20].
2.1.5. Surface-induced aggregation (Mechanism 5)
Finally, Mechanism 5 describes a surface-induced aggregation
process, in which native proteins first adsorb to an interface, after
which they undergo conformational changes or partial unfolding
(Fig. 1, scheme 5). The resulting non-native conformation then serves
as a starting point for aggregation in solution or directly on the
surface, as described in Mechanism 2 (above). While the previous
mechanisms have dealt with proteins in solution, this “heteroge-
neous” mechanism requires the presence of an interface (typically air-
water or solid-water). Protein binding at the air-water interface can
be attributed to hydrophobic interactions, while electrostatic in-
teractions often contribute at the solid-liquid interface. Nonionic
surfactants are used in this case to protect and stabilize proteins
against surface activity loss and/or surface-induced aggregation,
either by binding to the proteins and preventing protein-protein
associations, or by saturating the interface and thus minimizing
adsorption and subsequent conformational changes. These effects will
be discussed in detail in Section 3, below.
The nonionic surfactants Tween 20® and Tween 80® were seen to
protect albutropin, a recombinant human growth hormone–albumin
fusion protein, against agitation-induced aggregation in solution [21].
Although the binding affinity between the protein and Tween® was
different for different Tween® formulations, aggregation was completely
inhibited by the surfactant binding directly to the protein, at concentra-
tions well below the critical micelle concentration (CMC). The
surfactants increased the protein conformational stability by increasing
the free energy of unfolding associated with denaturation/aggregation.
Joshi et al. investigated the stabilization of non-agitated samples of
human recombinant Factor VIII (rFVIII) against aggregation in the
presence of polysorbate 80. Association of the rFVIII with the
surfactant in solution provided an effective steric barrier to aggrega-
tion, although shear fields were found to interfere with the stability of
the polysorbate 80-rFVIII association. At high concentrations of
polysorbate 80, however, the enhanced stabilization of agitated rFVIII
was attributed to rapid and preferential adsorption of polysorbate 80
at nascent air-water interfaces [22].
The extent of aggregation also depends upon the surface chemistry
of the container in which the protein is stressed. For example, more
aggregation was observed in Teflon®-like containers than in glass
containers after a freeze-thaw cycle [23]. In another study, rFVIII was
adsorbed on colloidal particles with hydrophilic or hydrophobic
surfaces and net positive or negative surface charge densities.
Hydrophilic surfaces exhibited relatively high rFVIII adsorption, but
did not induce large changes in structure or biological activity. In
contrast, exposure to hydrophobic nanoparticles caused substantial
changes in tertiary structure and reduced the biological activity (as
1164 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171

measured by activated partial thromboplastin time) of rFVIII. High sold commercially as Tween 20®, and polysorbate 80 (polyox-
surfactant concentrations, however, reduced these surface-induced yethylene sorbitan monooleate, or Tween 80®).
effects due to competitive hindrance of rFVIII adsorption at the Polysorbate 80 is considerably more surface-active and has a lower
surfactant-coated surface [24]. CMC than polysorbate 20, because it has a longer and monounsatu-
rated aliphatic chain [28]. Polysorbate 80 also exhibits a weaker
interaction with human serum albumin than polysorbate 20, again
2.2. Surfactants used in drug products
due to its longer fatty acid chain [21,27]. Differences in the binding
affinity and interaction of polysorbate 20 and 80 with darbepoetin alfa
Surfactants are amphipathic, surface-active molecules that readily
have been reported. Polysorbate 80 binds to darbepoetin alfa with
adsorb at interfaces. Although literally thousands of different
minimal effect on its tertiary structure, while binding of polysorbate
surfactants are commercially available, all generally consist of a
20 binding caused partial unfolding of the protein [29].
hydrophilic “head”, which can be ionic or a highly polar polymer, and
As mentioned above, polysorbates are widely reported to
a hydrophobic “tail”, often a long-chain aliphatic hydrocarbon group.
suppress aggregation upon agitation, shaking, freeze-drying and
Surfactants can be classified as anionic, cationic, nonionic and
freeze-thawing processes, and can substantially prevent protein
amphoteric based upon the nature of the hydrophilic “head”. An
adsorption at solid surfaces [21,24,30–33]. However, the effective-
excellent example of a widely-used anionic surfactant is sodium
ness appears to depend on the particular stress involved: in one
dodecyl sulfate (SDS); the dodecyl (C12) tail is hydrophobic, while the
study, stirring of an antibody suspension was found to be more
sulfate group is highly polar. Surfactants have wide-spread applica-
stressful than shaking, despite the renewal of air-water interfaces
tions in industry as emulsifiers, foaming agents, wetting agents,
during shaking. Polysorbate 20 at concentrations above 0.0025%
dispersants and detergents. The pharmaceutical and biotechnology
(w/v) inhibited aggregation during shaking. However, at lower
industries primarily use nonionic surfactants for a variety of
polysorbate concentrations, the protein was destabilized by
applications (including stabilization of protein therapeutics), because
shaking, and much higher surfactant concentrations were required
these surfactants exhibit low toxicity and low sensitivity to the
to stabilize stirred suspensions [34]. The polysorbates are suscep-
presence of electrolytes.
tible to autoxidation at moderate temperatures, primarily by radical
At low concentrations, surfactants will adsorb to all available
reactions at the PEO and unsaturation sites of the olefinic moieties,
interfaces, replacing the higher energy molecules, and lowering the
and hydrolysis was observed as a significant mechanism of
overall interfacial free energy of the system. However, as more
degradation at higher temperatures [35]. In another study, addition
surfactant molecules are introduced, eventually the interfaces become
of Tween 80® inhibited aggregation of IL-2 mutein during shaking.
saturated. At this point, energy reduction is achieved by formation of
Paradoxically, Tween 80® accelerated the aggregation of the same
micelles or other aggregated states, in which the hydrophobic “tails”
protein in a temperature-dependent manner during storage. The
are in the center and away from the surrounding water. The critical
build-up of peroxides from autoxidation of degraded polysorbates
micelle concentration (CMC) is defined as the bulk concentration of
increased the oxidization rate of the protein, therefore compromis-
surfactant at which micellization begins to occur, and is an important
ing its stability in storage [36].
fundamental property of a surfactant.
However, the CMC does not completely describe the surfactant's
2.2.2. Poloxamers
effect in a protein mixture. If the surfactant has a high affinity for a
Triblock copolymers of the form polyethylene oxide–polypropylene
surface, then surfactant concentrations near the CMC will tend to
oxide–polyethylene oxide (PEO–PPO–PEO), or poloxamers (commer-
stabilize protein against surface-induced denaturation, even when no
cially available as Pluronics® or SynperonicsTM), constitute another
specific binding of the surfactant to the protein is observed. In
class of nonionic surfactant commonly used in the pharmaceutical
contrast, if the surfactant stabilizes proteins by directly binding to
industry (Fig. 3). The poloxamers are listed as pharmaceutical excipients
them, the effective surfactant concentration is related to the ratio of
in the U.S. and British Pharmacopoeia, and have been used extensively in
surfactant to protein, rather than the CMC [25]. Equilibrium air-water
a variety of pharmaceutical formulations [37].
interfacial tensiometry measurements of surfactant solutions at
Poloxamers show complex aggregation behavior, involving unimers,
various concentrations are commonly used to estimate the CMC. In
oligomers, micelles of various geometries, and larger clusters, with strong
this approach, the CMC is determined as the bulk surfactant
temperature and concentration dependences. The critical micelle
concentration beyond which the equilibrium interfacial tension is
temperature and CMC values of such triblocks have been estimated
independent of surfactant concentration (i.e. adding more surfactant
over a wide range of molecular weights and PPO/PEO ratios, by a number
has no effect on the interfacial tension). This approach is often applied
of different methods [26,38–41]. In general, triblocks with a larger
to identify the apparent CMC of a surfactant in protein-surfactant
hydrophobic (PPO) domain form micelles at lower concentrations or, at a
mixtures as well. In either case, it is implicitly assumed that when the
constant triblock molar concentration, have lower critical micelle
CMC of the surfactant is met, the steady-state interfacial tension is
temperatures. For a given PPO:PEO ratio, triblocks of higher molecular
governed entirely by the surfactant at the interface, and independent
weight form micelles at lower concentrations and temperatures. The size
of other factors. It is important to note that experimental measures of
of the hydrophilic PEO group appears to play a smaller role in the
the CMC are generally not sharp transitions, and are strongly
micellization process. Alexandridis et al. [39] performed a thermody-
dependent upon factors such as ionic strength and temperature.
namic analysis of the formation of triblock micelles, to obtain standard
Thus, literature values for a given surfactant may vary widely between
free energies, enthalpies, and entropies of micellization for a number of
reports [26].
poloxamers. They found that the standard enthalpy of micellization was
positive for all triblocks tested, indicating that the transfer of unimers
2.2.1. Polysorbates from solution to the micelle is an enthalpically unfavorable, endothermic
Polysorbates have a common structure consisting of a sorbitan process. A negative entropy contribution (resulting from removal of the
ring with poly(ethylene oxide) at the hydroxyl positions, and differ highly-ordered clathrate cages of water molecules that surround a
only in the structures of the fatty acid side chains (Fig. 2). nonpolar molecule in an aqueous environment) was thus implicated as
Differences in the length and unsaturation of the fatty acid side the driving force for micellization.
chain structures cause the binding affinities of polysorbates with Poloxamer 188 (BASF Pluronic® F68, Fig. 3) is widely used for the
proteins to differ [27]. The most commonly-used nonionic surfac- large scale production of mammalian cell culture, and also where
tants are polysorbate 20 (polyoxyethylene sorbitan monolaureate), bioreactors are increasingly used to amplify a cell population. It is
 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171
Fig. 2. Chemical structure of polysorbate surfactants. The aliphatic (hydrophobic) tails polysorbate 20 and 80 vary in length and degree of unsaturation, while the PEO content
remains constant.
used as a shear-protective excipient to enhance cell yield in agitated
cultures and reduce cell adhesion in stationary cultures [42]. Two
mechanisms have been proposed in the literature to explain the cell
protection effect of poloxamer 188. One suggests that it affects the
culture medium characteristics, by inhibiting damage associated with
cell-bubble interactions when, for example, a bubble breaks at the air-
water interface. Another suggests that cells exhibit higher resistance
to shear stress in the presence of the triblocks. Poloxamer 188 has also
been reported to facilitate the refolding and to suppress aggregation
of a thermally denatured protein [43]. Removal of poloxamer 188
during product recovery may compromise the product yield, as well
as inhibit the growth of some cell lines [44].
2.3. Surfactant modulation of protein adsorption and aggregation
Surfactants stabilize proteins by two major mechanisms: (a) by
preferentially locating at an interface, in this way precluding protein
adsorption, and/or (b) by associating with proteins in solution, in this
way stabilizing them against close approach and inhibiting aggregation
(Fig. 4). Some surfactants may function according to only one of these
mechanisms, while others may function according to both.
2.3.1. Protein stabilization by surfactant adsorption at interfaces
A number of experimental investigations of the interfacial
behavior of surfactant and protein mixtures have been conducted,
and these have identified three possible adsorption outcomes:
complete hindrance, reduced amounts, or increased amounts of
protein adsorption. Complete hindrance is attributed to the faster
diffusion of the (generally smaller) surfactant molecules to the
interface, as compared to the much larger protein molecules. The
adsorbed surfactant layer coats the interface, and sterically prevents
protein adsorption. Reduced or increased amounts of adsorption are
usually attributed to the formation of surfactant-protein complexes
with reduced or increased surface affinity, respectively. In either case,
the behavior of these complexes is considerably different from that of
the pure protein or surfactant in solution.
An important goal in biotechnology process development and
biopharmaceutical formulation engineering is to minimize the protein
loss that occurs throughout the process by colloidal and interfacial
mechanisms, e.g., aggregation and adsorption [45,46]. In order to
achieve this, a fundamental understanding of the mechanisms
underlying surfactant effectiveness is necessary. In particular, better
understanding of the specific roles of the surfactant, protein, and
Fig. 3. Chemical structure of the PEO-PPO-PEO triblock copolymer Poloxamer 188.
Similar products with various molecular weights and PEO:PPO ratios are also
commercially available [40].
1165
surfactant-protein complex in modulating interfacial behavior will
provide direction for much-needed process improvements in the
production and finishing of therapeutic proteins.
2.3.1.1. Expectations based on sequential and competitive adsorption
experiments. The sequential introduction of a surfactant following
protein adsorption at an interface may result in the removal of
adsorbed protein, due to the formation of surfactant-protein com-
plexes and subsequent solubilization of these complexes. Alterna-
tively, adsorbed protein may be displaced by surfactant on account of
a stronger surfactant-surface association. The extent of surfactant-
mediated removal of adsorbed protein depends on protein, surfactant
and surface properties, and also other factors [47]. In general, the
difference in the amount of adsorbed protein eluted by anionic,
cationic and nonionic surfactants correlates with the strength of
binding between the surfactant and the protein in solution [48].
Nonionic surfactants, which are known to bind rather weakly to
proteins, are least effective in removing adsorbed protein molecules
from the interface. In particular, when introduced to an adsorbed
protein layer on a hydrophilic surface, nonionic surfactants generally
have little effect on the adsorbed amount. In contrast, on hydrophobic
surfaces, nonionic surfactants typically have a substantial effect on the
adsorbed protein, presumably because of the difference in surfactant
binding strength at the interface [49].
Joshi and McGuire [50] have described the interaction of lysozyme,
a well-characterized globular protein, with the nonionic surfactant
polysorbate 80 at solid-water interfaces. The concentration of the
surfactant, as well as the method of surfactant and protein
introduction to the surface (i.e. sequential or combined) was varied
in order to elucidate the separate roles of protein, surfactant, and the
protein-surfactant complex in determining adsorption outcomes.
They reported a decrease in lysozyme adsorption on hydrophobic
silica upon addition of polysorbate 80, and this reduction in adsorbed
protein increased with the concentration of polysorbate 80 in
solution. Sequential adsorption experiments showed that, at suffi-
ciently high concentration, polysorbate 80 was able to remove
adsorbed lysozyme from a hydrophobic surface. In addition, if
polysorbate 80 was introduced to the hydrophobic surface prior to
addition of lysozyme, adsorption of the protein was reduced or even
eliminated. On the other hand, polysorbate 80 had no effect on the
adsorption of lysozyme onto hydrophilic silica. Finally, sequential
adsorption experiments showed that polysorbate 80, when intro-
duced to the interface either before or after adsorption of lysozyme,
had no effect on the amount of lysozyme adsorbed. The observed
differences in protein adsorption were attributed to surface-depen-
dent differences in the binding affinity of polysorbate 80 to
hydrophobic or hydrophilic surfaces; this work emphasizes the
importance of direct interactions between the surfactant and the
solid surface, relative to surfactant-protein interactions in solution.
Accordingly, the rapid diffusion of the small surfactant molecules to
the interface (relative to proteins) is likely to contribute to a reduction
in protein adsorption only if the surfactant-surface affinity is suf-
ficiently high.
1166 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171

Fig. 4. Mechanisms of stabilization of proteins by surfactants, which may (a) dominate the interface and prevent protein adsorption, or (b) preferentially associate with proteins and
thus prevent close approach and aggregation.

2.3.1.2. On the stabilization of rFVIII by polysorbate 80 at solid-water
interfaces. Lysozyme is a much-used “model” protein for the study of
adsorption phenomena in a number of well-controlled circumstances,
but results of such work have contributed to forming a foundation for
the greater understanding of the behavior of more complex
therapeutic proteins in surfactant-containing formulations. The
adsorption, structural alteration and biological activity of a recombi-
nant Factor VIII (rFVIII) was investigated at a hydrophilic and
hydrophobic solid-water interface in the presence of polysorbate 80
[24]. As in the case of polysorbate 80 and lysozyme, association of
polysorbate 80 and rFVIII in solution was observed to be entirely
ineffective in reducing rFVIII adsorption, indicating that the surfactant
prevents adsorption by coating the interface, not the individual
protein molecules. Moreover, observations were attributed to the
high surfactant binding strength at the hydrophobic relative to the
hydrophilic surface.
In the absence of surfactant, proteins can be expected to adsorb
with high affinity to hydrophobic surfaces as well as negatively-
charged, positively-charged, and electronically neutral surfaces [51].
Substantial reductions in protein adsorption can be observed with
surfactant addition under appropriate circumstances, or in general
through the application of so-called “nonfouling” coatings, such as
those exhibiting pendant PEO chains [52–55]. The pendant polymer
chains resist protein adsorption by several mechanisms, primarily
steric repulsion [56]. It is thus reasonable that steric repulsion is a
requirement for eliminating protein adsorption, and explains the
nonfouling effect of such coatings. In the context of the work
summarized in relation to polysorbate 80, steric repulsion adequately

Fig. 5. Schematic illustration of surface tension depression associated with five regimes of protein-surfactant and surfactant-interface interactions. Redrawn with permission from [58].
 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171
explains the protein-repellent effect of added surfactants in the
presence of surfaces for which surfactant-surface binding is strong,
and the absence of any significant effect of added surfactant with
surfaces for which the surfactant-surface binding is weak. It follows
that steric repulsion is a requirement for the surfactant-mediated
prevention of protein aggregation.
2.4. Protein stabilization by surfactant-protein association
Protein stabilization by association of surfactants requires only
sufficiently strong surfactant-protein interaction, and would be
effective in reducing protein adsorption, regardless of the strength
of surfactant-surface binding. It is instructive to consider this
mechanism of protein stabilization, with reference to results of
surfactant action recorded at air-water interfaces in the presence and
absence of proteins.
2.4.1. A simple view of surfactant-protein mixtures at the air-water
interface
The molecular dynamics contributing to changes in air-water
interfacial tension for protein-surfactant mixtures are complex, but
offer an insight into the mechanisms of surfactant interactions with
interfaces and proteins. In ideal circumstances (i.e., for random chain
protein molecules and small, ionic surfactants) the following
equilibrium behavior is expected with increasing surfactant concen-
tration (Fig. 5) [57,58]:
Region 1. At very low surfactant concentrations, the steady-state
interfacial tension is the same as it would be for a pure protein
solution. The relatively few surfactant molecules have little or no
effect on surface tension.
Region 2. As surfactant concentration increases, the interfacial
tension decreases, due to surfactant occupation of “empty sites” at
the air-water interface, as well as the formation of surface-active
protein-surfactant complexes.
Region 3. At higher surfactant concentrations, the interfacial tension
is expected to plateau, presumably because it is energetically
favorable for surfactant to bind to protein at these concentrations
(in this range, the CMC recorded for the surfactant in protein-free
buffer may be exceeded).
Region 4. Equilibrium interfacial tension decreases again with
increasing surfactant concentration, as a result of complete displace-
ment of protein from the interface by the surfactant.
Region 5. Further increases in surfactant concentration have no effect
on interfacial tension, and a second plateau is reached. In this regime,
the CMC, which is specific to the protein concentration used in the
experiment, has been reached, and no further surfactants can adsorb
to the air-water interface [59].
This description relates to an idealized protein-surfactant mixture
at equilibrium, and is a useful reference for interpreting observations of
systems of greater complexity. However, for theoretical and practical
reasons, measurements of the true interfacial equilibrium are usually
not possible for real protein-surfactant mixtures. This is due mainly
to the inherent irreversibility of protein adsorption, as well as
uncertainties in the measurements required by the experiments.
The interfacial tension kinetic and steady-state behaviors exhib-
ited by surfactant solutions, as a function of surfactant concentration,
and in the presence and absence of protein, have been recorded for a
number of systems. Comparison of the steady-state surface tension
recorded for surfactant-protein mixtures with that of surfactant alone
(at similar concentrations) can be used to reveal whether protein
adsorption is evident, or if the steady-state interfacial behavior is
1167
governed entirely by surfactant. In particular, if no appreciable
difference is recorded between the steady-state value of interfacial
tension demonstrated by protein-surfactant mixtures and by the
surfactant alone at a similar concentration, one may tentatively
conclude that only the surfactant undergoes appreciable adsorption at
the interface (Fig. 6). For example, air-water interfacial tensiometry
experiments were performed with mixtures of rFVIII and polysorbate
80. The measured steady-state interfacial tensions, with and without
the protein, were identical at surfactant concentrations above 18 ppm,
indicating that the surface was dominated by the surfactant [22].
3. A testable, thermodynamic argument to guide surfactant selection
3.1. Insights gained from intact and protein-depleted pulmonary
surfactant
Schram and Hall [60] determined the influence of the two
hydrophobic proteins, SP-B and SP-C, on the thermodynamic barriers
that limit the adsorption of pulmonary surfactant vesicles to the air–
water interface in the lung. Vesicle adsorption, in this case, is
characterized by separation of the surfactant acyl chains, followed
by fusion of the bilayer vesicle with the interface to form a monolayer
(Fig. 7).
For this purpose they measured the kinetics of adsorption (based
on interfacial tensiometry) for intact calf lung surfactant extract, and
compared them with adsorption of an extract containing the complete
set of surfactant lipids, but depleted of the SP-B and SP-C proteins. The
surfactant proteins SP-B and SP-C are critical for normal respiration,
and accelerate the adsorption of intact surfactant (relative to protein-
free surfactant) more than ten-fold. This physiological behavior was
accurately reflected in the surface tension kinetic results recorded by
Schram and Hall. They interpreted their kinetic results for intact and
protein-free surfactant adsorption with reference to a mechanism for
vesicle adsorption. They postulated that vesicle adsorption and fusion
is governed by the formation of a rate-limiting structural intermediate
between the free and adsorbed forms (Fig. 7).
In particular they measured the rate constant (km) characterizing
the slope of the surface tension–time isotherm during the initial
decrease in surface tension, at each of a series of different
temperatures and concentrations, according to:
n
rate = km ⋅ c ; ð1Þ
where n, the order of the reaction, was obtained from measurements
of the initial adsorption rate at each concentration, c, in the bulk
phase.
Fig. 6. Schematic of differences in kinetics and extent of surface tension depression by
surfactant alone (S) or surfactant with protein (S + P), at different surfactant
concentrations ([S]) for a constant protein concentration. In general, surface tension
depression increases faster at a given [S] in the presence of protein. Steady-state surface
tensions corresponding to S and S + P converge at sufficiently high [S].
1168 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171
The activation energies, Ea, for adsorption were then derived from
the slopes of plots of the experimental ln km vs. 1/T data, according to
the Arrhenius equation:
 
Ea 1
ln km = − ⋅ + ln A;
R T
where R is the gas constant, T is temperature, and A is the Arrhenius
pre-exponential factor. They invoked transition-state theory to
consider the expected effect of temperature, in terms of an
equilibrium between the “reactants” (i.e., the vesicles and unoccupied
air-water interface) and an activated complex. From this model, the
rate constant can be described in thermodynamic terms:
 
kb T −ΔG = RT
km = e ;
h surfactant aggregate are based on entropy. Thus a thermodynamic
where kb and h are Boltzmann's and Planck's constants, respectively, and
ΔG is the Gibbs free energy of transition. Thus, since ΔG = ΔΗ− ΤΔS,
     
km ΔH 1 k ΔS
ln =− ⋅ + ln b +
T R T h R
The slope and intercept of plots of ln km/T vs. 1/T therefore provide
quantitative estimates of the enthalpy (ΔH) and entropy (ΔS) of the
transition.
The transition of a bilayer to form an interfacial monolayer requires
a transient exposure of the hydrophobic tails of the surfactant lipids to
the aqueous environment (Fig. 7), and consequently has an unfavorable
entropy of transition. Schram and Hall's analysis, however, showed
that the surfactant proteins did not affect the entropy of transition;
rather, the essential effect of the proteins was to minimize an
unfavorable enthalpy barrier to formation of the structural intermedi-
ate. This enthalpic cost was attributed to the dissociation of the
surfactant acyl chains during the separation of the leaves of the bilayers.
An interesting observation characteristic of surface tension
depression by surfactant-protein mixtures is that the kinetics of
surface tension depression in such mixtures tend to be uniformly
greater than that recorded for surfactant alone at the same
concentration, regardless of whether the final steady-state surface
tension is similar in each case (Fig. 6). This kind of “synergistic” effect
is well documented for synthetic polymer-surfactant mixtures [61],
but less well understood in relation to protein-surfactant mixtures.
Joshi et al. [22] found the rate of surface tension decrease to be greater
for polysorbate 80-rFVIII mixtures than for polysorbate acting alone,
at all polysorbate concentrations studied in that work (8 to 108 ppm).
Fig. 7. Schematic of the rate-limiting intermediate state in the transition from a
phospholipid bilayer to an interfacial monolayer. Proteins located within the bilayer
were found to reduce the enthalpic barrier of the intermediate state. Adapted from [60]
with permission from Elsevier.
The reasons for this have not been articulated in any quantitative
fashion, but might be explained in part using an approach to the
problem similar to that outlined by Schram and Hall [60]. This
approach might also provide direction for surfactant selection (or
surfactant design) to more effectively manage issues surrounding
ð2Þ
aggregation and adsorption loss.
3.2. Surfactant-protein association at surfactant concentrations above
the CMC
Consider the case of surfactant adsorption in the presence of
protein, at surfactant concentrations above the CMC (or otherwise
consistent with the presence of associated unimers, if the surfactant
system is not governed by any obvious CMC). The hydrophobic
interactions that maintain the structure of a micelle or other
ð3Þ
analysis similar to that used by Schram and Hall [60] can be applied to
the notion of protein-mediated acceleration of surfactant adsorption
introduced in Section 3.1. We suggest that proteins may accelerate the
adsorption of surfactants by reducing the major entropic barrier faced
by the surfactant in moving from the aggregate to interface, and/or by
ð4Þ
reducing the enthalpy of activation. In the latter case, the proteins
might destabilize the surfactant self-association, or they could
produce a “catalytic” reduction in the enthalpy of some structural
intermediate between unbound surfactant aggregates and adsorbed
surfactant unimers (Fig. 8). In either case, the likely source for a
change in enthalpy would be an alteration of the van der Waals
interactions among the regions on the surfactant molecules mediating
their self-association. Separation of surfactant monomers from
aggregates and their location at the air-water interface would disrupt
van der Waals interactions and produce an unfavorable enthalpy.
These arguments are similarly significant in relation to associa-
tions of large-molecule surfactants. The mechanism of adsorption and
the adsorption kinetics exhibited by poloxamers, for example,
strongly depends on their solution concentration during adsorption
[62,63]. At low concentrations, the triblocks exist as individual
molecules (unimers), and their adsorption may not uniformly cover
the entire available surface. At high concentrations, however,
adsorption is dominated by micelles or other aggregates, and the
PEO chains may inhibit the association between the hydrophobic
center blocks and the surface. Adsorption of poloxamers is slow in
comparison to small molecule surfactants, and any synergistic
“chaperone” effects on the kinetics of surface tension depression
(described in Section 2.4.1 and Fig. 6) are less apparent for mixtures of
selected proteins and, for example, poloxamer 188. We may
tentatively explain this behavior by noting that the strong surfac-
tant-protein associations in solution are not conducive to an enhanced
“delivery” of surfactant molecules to the interface (Fig. 8).
3.3. Surfactant-protein association at surfactant concentrations below
the CMC
There is convincing evidence that poloxamers interact with the
plasma membranes of cells. More hydrophobic poloxamers generally
show greater tendencies to incorporate into the cell plasma
membrane [63]. Gigout et al. [64] examined the incorporation of
poloxamer 188 into the cell plasma membrane and its subsequent
uptake by chondrocytes and CHO cells. They were able to conclusively
demonstrate that the triblocks did in fact enter the cells, and possibly
accumulate in the endocytic pathway.
While poloxamer 188 shows a high affinity for entropically-driven
associations with cell membranes, it is not expected to form micelles
in water except at very high concentrations [38,40,41,65]. It is
possible that the observed high affinity for cell surfaces can be
explained by the significantly decreased solubility of the surfactant in
the presence of salts. Patel et al. studied the micellization of a very
 H.J. Lee et al. / Advanced Drug Delivery Reviews 63 (2011) 1160–1171 1169

Fig. 8. Hypothetical mechanisms for transport of surfactant from an aggregated state to the surface. In the absence of protein (top), surfactant unimers must dissociate and migrate
through the liquid to the interface. Proteins may promote formation of a structural intermediate between surfactant aggregates and adsorbed unimers, reducing the thermodynamic
barriers associated with their location at the interface (bottom).

hydrophilic poloxamer (80% PEO) in various sodium salt solutions. even when the interface is only partially occupied by surfactant. Further
While the triblock did not form micelles in pure water at ambient research into these mechanisms is clearly warranted.
temperatures, it exhibited aggregation and micellization in salt
solutions. The cloud point and critical micelle temperature (CMT)
4. Conclusions
decreased substantially in the presence of salts. They found that the
average micelle size increased with salt concentration, and the
Surfactants stabilize proteins by their preferential location at an
relative effect of the different anions to enhance micellization
interface and/or their association with protein in solution. In the
followed the Hofmeister series (i.e. PO43– N SO42– N F – N Cl – N Br –), con-
common case of surfactant-protein interaction governed by hydropho-
sistent with salting out of the poloxamer [66].
bic association, the molecular origins of surfactant-mediated stabiliza-
Clouding is a thermodynamic phase transition that is characteristic
tion of protein can be inferred, in part, from quantitative models. Insights
of molecules containing PEO or other polyethers, and is commonly
can be gained by comparison of thermodynamic barriers that limit
associated with triblocks and other nonionic surfactants in water. It is
surfactant adsorption, from surfactant-protein mixtures and protein-
due in part to changes in hydrogen bonding and increasingly
free surfactant solutions, to the air–water interface. These barriers are
attractive interactions between the PEO chains as the temperature
defined by the enthalpy and entropy of transition from free (whether
increases, making the water a “less good” solvent [67]. The influence
unimeric or aggregated) surfactant molecules to adsorbed surfactants,
of salts can be explained in a similar fashion, being primarily caused
via formation of a rate-limiting structural intermediate. This is easily
by the existence of a salt-deficient zone surrounding the PEO chains:
done, for example, through analysis of surface tension kinetic data
small ions with little polarizability would be repelled by the poorly
recorded at different temperatures. Concentration effects on the
polarizable PEO chain. This salt-deficient zone gives rise to an
aggregation state of a surfactant, effects of salt on micellization, and
attractive component in the interaction between PEO segments. As
the thermodynamic barriers to its adsorption are particularly important
polymer segments approach each other, their salt depletion zones
factors in determining the effectiveness of a given surfactant at
overlap and the surrounding water molecules are liberated to the bulk
stabilizing a protein. A fundamental understanding of the mechanisms
solution. The excluded water molecules have a lower chemical
of aggregation and how surfactants interact with interfaces and proteins
potential in the bulk, a thermodynamically favored state [38].
(particularly the preferential location of a surfactant at an interface, or its
Cell surfaces (as well as the “surfaces” of proteins) are charged and
association with protein in solution), provides guidance in selecting
“separated” from the bulk solution by a surrounding ion-enriched
surfactants and excipients to reduce protein losses in a given application.
electrical double layer [68,69]. It is reasonable to anticipate a marked
decrease in solubility of a poloxamer at the vicinity of such a colloidal
surface, owing to the higher salt concentration at the interface than in Acknowledgments
the bulk. It is thus tempting to hypothesize that a surfactant unimer
that approaches a cell or protein “surface” would find itself in a region The authors are grateful to David Brems and Sekhar Kanapuram at
of higher ionic strength, and be driven to associate with the surface by Amgen for helpful discussions during the preparation of this
a “salting out” mechanism. Thus, surfactants could effectively stabilize manuscript.
a protein by association with the surface, forming a surfactant-protein
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