Advanced Drug Delivery Reviews -...

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Advanced Drug Delivery Reviews xxx (2011) xxx–xxx

ADR-12165; No of Pages 12

Contents lists available at ScienceDirect

Advanced Drug Delivery Reviews

j ourna l homepage: www.e lsev ie r.com/ locate /addr

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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 97331b Process and Product Development Department Amgen Inc., Thousand Oaks, CA 91320

☆ This review is part of the Advanced Drug Delivery Re⁎ Corresponding author at: School of Chemical, Biolog

6306; fax: +1 541 737 4600.E-mail address: [email protected] (J. McGuire)

0169-409X/$ – see front matter © 2011 Published by Edoi:10.1016/j.addr.2011.06.015

Please cite this article as: H.J. Lee, et al., Moldoi:10.1016/j.addr.2011.06.015

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Article history:Received 30 March 2011Accepted 29 June 2011Available online xxxx

Keywords:AdsorptionAggregationFormulationProteinStabilizationSurfactant

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production, formulation and administration of therapeutic proteins. Surfactants are commonly used inupstream and downstream processing and drug formulation. However, the effectiveness of a surfactantstrongly depends on its mechanism(s) of action and properties of the protein and interfaces. Surfactants canmodulate adsorption loss and aggregation by coating interfaces and/or participating in protein-surfactantassociations. Minimizing protein loss from colloidal and interfacial interaction requires a fundamentalunderstanding of the molecular factors underlying surfactant effectiveness and mechanism. These conceptsprovide direction for improvements in the manufacture and finishing of therapeutic proteins. We summarizethe roles of surfactants, proteins, and surfactant-protein complexes in modulating interfacial behavior andaggregation. These events depend on surfactant properties that may be quantified using a thermodynamicmodel, to provide physical/chemical direction for surfactant selection or design, and to effectively reduceaggregation and adsorption loss.

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views theme issue on “Formulating Biomolecules: Mechical and Environmental Engineering, Oregon State Univer

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lsevier B.V.

ecular origins of surfactant-mediated stabiliza

© 2011 Published by Elsevier B.V.

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2. Managing protein aggregation and adsorption loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1. Mechanisms of aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1.1. Concentration-induced aggregation (Mechanism 1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.2. Aggregation induced by conformational change (Mechanism 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.3. Aggregation induced by chemical reaction (Mechanism 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.4. Nucleation-dependent aggregation (Mechanism 4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.5. Surface-induced aggregation (Mechanism 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.2. Surfactants used in drug products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2.1. Polysorbates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2.2. Poloxamers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.3. Surfactant modulation of protein adsorption and aggregation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.3.1. Protein stabilization by surfactant adsorption at interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.4. Protein stabilization by surfactant-protein association . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.4.1. A simple view of surfactant-protein mixtures at the air-water interface . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

3. A testable, thermodynamic argument to guide surfactant selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.1. Insights gained from intact and protein-depleted pulmonary surfactant. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.2. Surfactant-protein association at surfactant concentrations above the CMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03.3. Surfactant-protein association at surfactant concentrations below the CMC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

anistics Insights in Molecular Interactions”.sity, 103 Gleeson Hall, Corvallis, OR 97331–2702. Tel.: +1 541 737

tion of protein drugs, Adv. Drug Deliv. Rev. (2011),

http://dx.doi.org/10.1016/j.addr.2011.06.015

mailto:[email protected]


http://www.sciencedirect.com/science/journal/0169409X


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2 H.J. Lee et al. / Advanced Drug Delivery Reviews xxx (2011) xxx–xxx

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1. Introduction

In recent years, the number of protein and peptide therapeuticsreaching the marketplace has increased significantly for most majorpharmaceutical 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 andmanipulation [1]. Since then, rapid developments in biotechnologyhave enabled the commercial production of various hormones, bloodfactors, cytokines, and fully human monoclonal antibodies. Suchtherapeutic 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, largemolecules that aremade up of awell-defined sequence of amino acids,andmay be produced through a combination of chemical or biologicalmeans. Proteins adopt distinct three-dimensional structures that areusually necessary for correct function, but which are often highlysensitive to the surrounding environment and may be easily distortedor altered. Although protein drugs are generally considered to havefewer inherent side effects than traditional chemical agents, they areusually very surface-active and are more susceptible to activity lossthrough adsorption, structural unfolding and aggregation than smallmolecules. This is a substantial problem for the biopharmaceuticalindustry, because losses of biological activity through aggregation orsurface-induced structural alteration (denaturation) are encounteredthroughout the production, formulation and administration oftherapeutic proteins. Therefore, considerable efforts have beenmade to identify the causes of adsorption loss and aggregation, andto develop effective methods to minimize these detrimental effectsand associated costs. Several mitigation strategies are used in thebiotechnology industries to stabilize therapeutic proteins, but theaddition of surfactants appears to be a general approach [2].

Proteins can often be stabilized against adsorption loss through theuse of properly-chosen surfactants. Preferential location of surfactantmolecules at interfaces, such as the walls of glass vials or the surfacesof bubbles, may strongly inhibit adsorption of proteins and preventtheir subsequent denaturation or loss. In addition, formation ofsurfactant-protein complexes in solution can reduce the surfaceactivity of the proteins, thus stabilizing them toward close approachand aggregation. Aggregation can also be inhibited by a variety of non-surfactant stabilizers, which are selected based on their ability toinhibit specific molecular mechanism(s) that govern aggregationphenomena in a given system. In Section 2, below, we briefly outlinethe major mechanisms that contribute to aggregation and surface-induced conformational changes. Some chemical strategies used toinhibit or slow thosemechanisms, including the use of surfactants, arealso presented. The stabilization of proteins by surfactants, in thepresence of interfaces, is discussed in Section 3. Particular emphasis isgiven to themechanisms bywhich complexes of protein and surfactantmolecules 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 studiedand reported extensively by academia, industry and regulatoryagencies. Aggregation is highly undesirable due to the profoundimpact on the stability of the drug product, which can result in loss ofactivity, unwanted immunogenic responses, and increased rate ofrejection as a marketable product [3]. Several workers have reportedon the different mechanisms of aggregation, and suggest possiblemethods to inhibit aggregation [4–6]. Various external chemical orphysical factors may be responsible. Additionally, the inherentproperties of the protein (e.g. charged or hydrophobic regions) maymake it unusually susceptible to aggregation. In such cases, ag-gregation can often be inhibited bymodifying themolecular properties

Please cite this article as: H.J. Lee, et al., Molecular origins of surfactant-mdoi:10.1016/j.addr.2011.06.015

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or by changing the external environment [7,8]. Inherent properties canbe effectively modified by site-directed mutagenesis [7] or chemicalmodification (e.g. PEGylation) [8], but such modifications maycompromise the biological activity of the protein. Thus, the simplestand most common method of inhibiting aggregation is to change thenature of the environment surrounding the protein by adjustingsolution conditions such as pH, or by adding stabilizers/excipients. Bycarefully examining themechanism(s) responsible for the aggregation,we can identify specific changes or stabilizing molecules that willeffectively 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, andproduces unwanted and detrimental effects on a therapeutic drugproduct. Aggregation occurs through several different major mecha-nisms and pathways (Fig. 1), discussed in detail in the examplesbelow. Although a particular mechanism may identify an aggregationpathway for a particular protein, it may not be relevant for anotherprotein. Also, more than one mechanism or pathway may be simul-taneously responsible for destabilizing a protein formulation [5]. Afundamental understanding of the mechanisms of aggregation isnot only valuable in identifying the cause of a problem, but also helpfulin suggesting methods to suppress aggregation to an acceptable level.It may be noted that aggregation may or may not lead to precipitationor 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 reversibleaggregates especially in high concentration formulations. Mechanism1 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 (possiblythrough disulfide interchange reactions) is one cause of this irrevers-ibility [5]. IgG antibodies have been observed to form reversible solubleaggregates in high concentration solutions. Electrostatic interactionsand hydrogen bonds contribute to the self-association of IgGmolecules.Hydrophobic patches in the Fc region of IgG are considered to be amajor factor in inducing aggregation at higher concentrations [10].

Human interleukin-1 receptor anatagonist (IL-1ra) is part of theIL-1/Fibroblast Growth Factor family of proteins with a predominantlyβ-strand secondary structure. It self-associates and aggregateswithout changes to secondary structure at high concentrations andelevated temperatures. This self-association induced aggregation wasattributed to a positively-charged Lys96 residue in the IL-1ra molecule.Aggregation affinity was dependent on the buffer ionic strength andthe type of anion. Phosphate anion was found to inhibit aggregationmore weakly than citrate or pyrophosphate at pH 6.5. Proteolyticremoval of an unstructured N-terminal region containing anotherlysine residue also substantially reduced the rate of self-association. Itwas proposed that the anions compete for cationic sites on theprotein, preventing the formation of cation-π interactions betweenprotein molecules. Based on pK values at 25 °C, citrate andpyrophosphate anions would have 2 to 3 times more ionized groupsthan phosphate at pH 6.5. The relative affinity of the anion binding tothe cationic site (and hence, inhibition of aggregation) was correlatedwith the number of ionizable groups at a given pH [7].

Insulin aggregation is generally considered reversible at roomtemperature near its isoelectric point. This can be attributed toelectrostatic interactions due to the marked charge anisotropy ofthe polypeptide. Addition of heparin, a highly charged polyanion,prevented aggregation at pH higher than 6 by binding to the positivedomains of insulin to form heparin-insulin complexes. Heparin was

ediated stabilization of protein drugs, Adv. Drug Deliv. Rev. (2011),


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Fig. 1. Schematic illustration of five commonmechanisms of aggregation. Multiple mechanisms may be at work in any given system. Adapted with permission from [5]. Copyright ©2009 Bentham Science Publishers.

3H.J. Lee et al. / Advanced Drug Delivery Reviews xxx (2011) xxx–xxx

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also able to dissociate aggregate particles of insulin, indicating that theassociation 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 thenative state. InMechanism 2, proteins aggregate after they go through aconformational change or partial unfolding (denaturation), which is therate-limiting step (Fig. 1, scheme 2). Interactions between thedenatured proteins are typically driven by hydrophobic associations,and are usually strong enough to be practically irreversible. Externalfactors likeheat and shear can induce theprotein into anon-native state,as is commonly observed with the proteins in egg whites.

Stability of interferon-tau (INF-tau), which is a novel type 1interferon, depended on the type of buffering agent, even when thepH and ionic strength were kept constant. At pH 7.0, INF-tau in Trisand phosphate buffers at elevated temperatures was observed toaggregate, with a substantial loss of tertiary structure and slightlyexpanded non-native conformation. However, samples containingfree histidine as a buffering agent suppressed thermally-inducedaggregation, and little loss of tertiary structure was observed at thesame pH. Detectable bindingwas 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 ofhistidine 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 recombinanthuman granulocyte colony stimulating factor (rhGCSF)were observedto aggregate under physiological conditions [13]. Added sucrose wasable to stabilize rhGCSF, and inhibited aggregation under stressedconditions. The thermodynamic stability of rhGCSF increased with theaddition of sucrose, which is preferentially excluded from the surfaceof the protein [14,15]. In this system, sucrose acted as a stabilizer byshifting the equilibrium to favor the native compact species ratherthan 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 asoxidation, deamidation, or disulfide scrambling (Fig. 1, scheme 3).Chemical changes may profoundly alter protein properties such assolvent accessibility of hydrophobic patches, reduction in electrostaticrepulsion due to modification of charged residues, or disruptions of



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the native structure that trigger unfolding. It is important to note thatchemically different species are not necessarily degradation products,but may be formed during normal production of a drug. Truncatedpeptides 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 activatingtranscription factor 5, which consists of a single α-helix and a singlecysteine residue, was observed to be dependent on intermoleculardisulfide bonds which stabilize the native structure. Reduction of thedisulfide bond resulted in the unfolding of the peptide and exposedhydrophobic regions, which resulted in aggregation of the protein [16].

A detailed structural characterization of the effects of methionineoxidation on the stability of the human IgG Fc region was studied.Oxidation of methionine can generate a repulsive interaction betweenthe side chains of methioninine residues in the CH2 and CH3 domains,and thus disrupt the native structure. Although Met residues in bothCH2 and CH3 domains were affected by oxidation, more structuralperturbations were observed in the CH2 domain. Therefore, anincrease in aggregation would be likely due to the structuralinstability of the CH2 domain of the Fc region. Since aggregation wasobserved only for highly oxidized proteins, addition of excipients suchas methionine or sodium thiosulfate that acts as oxygen scavengerswould be sufficient in preventingmethionine oxidation that can causeaggregation [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 initiatedwhen a “critical nucleus” is formed in solution, and native proteinsare recruited, and often partially unfolded, to form aggregated species(Fig. 1, scheme 4). The process is not unlike the growth of a largecrystal from a supersaturated solution after addition of a seed crystal.In this case, the “seed” is a microscopic aggregated particle of adenatured or otherwise non-native conformation. A “lag phase”(often weeks or months) is characteristic of this mechanism. Duringthis lag phase (which may vary considerably from sample to sample),the seed nucleus grows, but no particles or precipitation can beobserved. After the formation of a critical nucleus, the aggregationprogresses rapidly, with the relatively sudden formation of visibleaggregates or precipitates in solution. These nuclei may be denaturedproteins, or solid contaminants (e.g. particles of silica from vials ormetal from pumps) [5].

An excellent example of this nucleation-dependent mechanism isthe 10-residue peptide of human amylin, which is used as a modelsystem to study self-assembly of amyloid fibril proteins. Amylin wasobserved to aggregate in response to low levels of asparaginedeamidation, such as might be found as impurities in syntheticamyloid peptides. Seeding solutions of the native peptide with smallamounts of deamidated peptide resulted in rapid aggregation to formcharacteristic amyloid structures. Additionally, when the affected sidechain of the deamidated peptide is deprotonated and negativelycharged (at physiological pH), electrostatic interactions with thepositively-charged N-terminus of another amylin peptide induce thepropagation of the aggregation event. A relevant point is thatphosphate anion is known to promote deamidation of Asp/Glnresidues [18].

Tungsten contamination from a needle tip was observed to inducesignificant protein aggregation in pre-filled syringes. Tungstenmicroparticles become soluble at lower pH, forming tungstenpolyanions 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 repulsionsbetween the native monomers to induce precipitation. However, atpH 6.0 and higher, tungsten polyanions do not form and aggregation


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was not observed. The authors caution that the small number oftungsten particles required to induce precipitation of the antibodies,combined with poor mixing in the needle, precluded defining anacceptable syringe volume for a given protein [19].

Silicone oil, a common lubricant in pharmaceutical applications,has also been implicated in aggregation of monoclonal antibodies inpre-filled syringes. Although silicone oil alone did not induceaggregation, a synergistic effect producing substantial aggregationwas observed when samples were agitated in the presence of siliconeoil. Perturbation of the monomeric state of the protein by acombination of air-water and oil-water interfacial stresses wasimplicated in the aggregation. Complete inhibition of silicone oil-induced protein loss was observed when the nonionic surfactantpolysorbate 20 was added (polysorbates are discussed in more detailin Section 2.2.1, below). Polysorbate 20 is known to compete withprotein molecules at air-water and oil-water interfaces (modelhydrophobic systems), where it prevents structural perturbationsand subsequent aggregation of the protein molecules at theunprotected 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, afterwhich they undergo conformational changes or partial unfolding(Fig. 1, scheme 5). The resulting non-native conformation then servesas a starting point for aggregation in solution or directly on thesurface, as described in Mechanism 2 (above). While the previousmechanisms 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 canbe attributed to hydrophobic interactions, while electrostatic in-teractions often contribute at the solid-liquid interface. Nonionicsurfactants are used in this case to protect and stabilize proteinsagainst surface activity loss and/or surface-induced aggregation,either by binding to the proteins and preventing protein-proteinassociations, or by saturating the interface and thus minimizingadsorption and subsequent conformational changes. These effects willbe discussed in detail in Section 3, below.

The nonionic surfactants Tween 20® and Tween 80® were seen toprotect albutropin, a recombinant human growth hormone–albuminfusion protein, against agitation-induced aggregation in solution [21].Although the binding affinity between the protein and Tween® wasdifferent for different Tween® formulations, aggregationwas completelyinhibited by the surfactant binding directly to the protein, at concentra-tions well below the critical micelle concentration (CMC). Thesurfactants increased the protein conformational stability by increasingthe free energy of unfolding associated with denaturation/aggregation.

Joshi et al. investigated the stabilization of non-agitated samples ofhuman recombinant Factor VIII (rFVIII) against aggregation in thepresence of polysorbate 80. Association of the rFVIII with thesurfactant in solution provided an effective steric barrier to aggrega-tion, although shear fields were found to interfere with the stability ofthe polysorbate 80-rFVIII association. At high concentrations ofpolysorbate 80, however, the enhanced stabilization of agitated rFVIIIwas attributed to rapid and preferential adsorption of polysorbate 80at nascent air-water interfaces [22].

The extent of aggregation also depends upon the surface chemistryof the container in which the protein is stressed. For example, moreaggregation was observed in Teflon®-like containers than in glasscontainers after a freeze-thaw cycle [23]. In another study, rFVIII wasadsorbed on colloidal particles with hydrophilic or hydrophobicsurfaces and net positive or negative surface charge densities.Hydrophilic surfaces exhibited relatively high rFVIII adsorption, butdid not induce large changes in structure or biological activity. Incontrast, exposure to hydrophobic nanoparticles caused substantialchanges in tertiary structure and reduced the biological activity (as



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measured by activated partial thromboplastin time) of rFVIII. Highsurfactant concentrations, however, reduced these surface-inducedeffects due to competitive hindrance of rFVIII adsorption at thesurfactant-coated surface [24].

2.2. Surfactants used in drug products

Surfactants are amphipathic, surface-active molecules that readilyadsorb at interfaces. Although literally thousands of differentsurfactants are commercially available, all generally consist of ahydrophilic “head”, which can be ionic or a highly polar polymer, anda hydrophobic “tail”, often a long-chain aliphatic hydrocarbon group.Surfactants can be classified as anionic, cationic, nonionic andamphoteric based upon the nature of the hydrophilic “head”. Anexcellent example of a widely-used anionic surfactant is sodiumdodecyl sulfate (SDS); the dodecyl (C12) tail is hydrophobic, while thesulfate group is highly polar. Surfactants have wide-spread applica-tions in industry as emulsifiers, foaming agents, wetting agents,dispersants and detergents. The pharmaceutical and biotechnologyindustries primarily use nonionic surfactants for a variety ofapplications (including stabilization of protein therapeutics), becausethese surfactants exhibit low toxicity and low sensitivity to thepresence of electrolytes.

At low concentrations, surfactants will adsorb to all availableinterfaces, replacing the higher energy molecules, and lowering theoverall interfacial free energy of the system. However, as moresurfactant molecules are introduced, eventually the interfaces becomesaturated. At this point, energy reduction is achieved by formation ofmicelles or other aggregated states, in which the hydrophobic “tails”are in the center and away from the surrounding water. The criticalmicelle concentration (CMC) is defined as the bulk concentration ofsurfactant at which micellization begins to occur, and is an importantfundamental property of a surfactant.

However, the CMC does not completely describe the surfactant'seffect in a protein mixture. If the surfactant has a high affinity for asurface, then surfactant concentrations near the CMC will tend tostabilize protein against surface-induced denaturation, even when nospecific binding of the surfactant to the protein is observed. Incontrast, if the surfactant stabilizes proteins by directly binding tothem, the effective surfactant concentration is related to the ratio ofsurfactant to protein, rather than the CMC [25]. Equilibrium air-waterinterfacial tensiometry measurements of surfactant solutions atvarious concentrations are commonly used to estimate the CMC. Inthis approach, the CMC is determined as the bulk surfactantconcentration beyond which the equilibrium interfacial tension isindependent of surfactant concentration (i.e. adding more surfactanthas no effect on the interfacial tension). This approach is often appliedto identify the apparent CMC of a surfactant in protein-surfactantmixtures as well. In either case, it is implicitly assumed that when theCMC of the surfactant is met, the steady-state interfacial tension isgoverned entirely by the surfactant at the interface, and independentof other factors. It is important to note that experimental measures ofthe CMC are generally not sharp transitions, and are stronglydependent upon factors such as ionic strength and temperature.Thus, literature values for a given surfactant may vary widely betweenreports [26].

2.2.1. PolysorbatesPolysorbates have a common structure consisting of a sorbitan

ring with poly(ethylene oxide) at the hydroxyl positions, and differonly in the structures of the fatty acid side chains (Fig. 2).Differences in the length and unsaturation of the fatty acid sidechain structures cause the binding affinities of polysorbates withproteins to differ [27]. The most commonly-used nonionic surfac-tants are polysorbate 20 (polyoxyethylene sorbitan monolaureate),


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sold commercially as Tween 20®, and polysorbate 80 (polyox-yethylene sorbitan monooleate, or Tween 80®).

Polysorbate 80 is considerably more surface-active and has a lowerCMC than polysorbate 20, because it has a longer and monounsatu-rated aliphatic chain [28]. Polysorbate 80 also exhibits a weakerinteraction with human serum albumin than polysorbate 20, againdue to its longer fatty acid chain [21,27]. Differences in the bindingaffinity and interaction of polysorbate 20 and 80with darbepoetin alfahave been reported. Polysorbate 80 binds to darbepoetin alfa withminimal effect on its tertiary structure, while binding of polysorbate20 binding caused partial unfolding of the protein [29].

As mentioned above, polysorbates are widely reported tosuppress aggregation upon agitation, shaking, freeze-drying andfreeze-thawing processes, and can substantially prevent proteinadsorption at solid surfaces [21,24,30–33]. However, the effective-ness appears to depend on the particular stress involved: in onestudy, stirring of an antibody suspension was found to be morestressful than shaking, despite the renewal of air-water interfacesduring shaking. Polysorbate 20 at concentrations above 0.0025%(w/v) inhibited aggregation during shaking. However, at lowerpolysorbate concentrations, the protein was destabilized byshaking, and much higher surfactant concentrations were requiredto stabilize stirred suspensions [34]. The polysorbates are suscep-tible to autoxidation at moderate temperatures, primarily by radicalreactions at the PEO and unsaturation sites of the olefinic moieties,and hydrolysis was observed as a significant mechanism ofdegradation at higher temperatures [35]. In another study, additionof Tween 80® inhibited aggregation of IL-2 mutein during shaking.Paradoxically, Tween 80® accelerated the aggregation of the sameprotein in a temperature-dependent manner during storage. Thebuild-up of peroxides from autoxidation of degraded polysorbatesincreased the oxidization rate of the protein, therefore compromis-ing its stability in storage [36].

2.2.2. PoloxamersTriblock copolymers of the form polyethylene oxide–polypropylene

oxide–polyethylene oxide (PEO–PPO–PEO), or poloxamers (commer-cially available as Pluronics® or SynperonicsTM), constitute anotherclass of nonionic surfactant commonly used in the pharmaceuticalindustry (Fig. 3). The poloxamers are listed aspharmaceutical excipientsin theU.S. andBritishPharmacopoeia, andhavebeenusedextensively ina variety of pharmaceutical formulations [37].

Poloxamers show complex aggregation behavior, involving unimers,oligomers,micelles of various geometries, and larger clusters,with strongtemperature and concentration dependences. The critical micelletemperature and CMC values of such triblocks have been estimatedover awide range ofmolecularweights and PPO/PEO ratios, by a numberof different methods [26,38–41]. In general, triblocks with a largerhydrophobic (PPO) domain formmicelles at lower concentrations or, at aconstant triblock molar concentration, have lower critical micelletemperatures. For a given PPO:PEO ratio, triblocks of higher molecularweight formmicelles at lower concentrations and temperatures. The sizeof the hydrophilic PEO group appears to play a smaller role in themicellization process. Alexandridis et al. [39] performed a thermody-namic analysis of the formation of triblock micelles, to obtain standardfree energies, enthalpies, and entropies of micellization for a number ofpoloxamers. They found that the standard enthalpy of micellization waspositive for all triblocks tested, indicating that the transfer of unimersfrom solution to themicelle is an enthalpically unfavorable, endothermicprocess. A negative entropy contribution (resulting from removal of thehighly-ordered clathrate cages of water molecules that surround anonpolar molecule in an aqueous environment) was thus implicated asthe driving force for micellization.

Poloxamer 188 (BASF Pluronic® F68, Fig. 3) is widely used for thelarge scale production of mammalian cell culture, and also wherebioreactors are increasingly used to amplify a cell population. It is



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used as a shear-protective excipient to enhance cell yield in agitatedcultures and reduce cell adhesion in stationary cultures [42]. Twomechanisms have been proposed in the literature to explain the cellprotection effect of poloxamer 188. One suggests that it affects theculture medium characteristics, by inhibiting damage associated withcell-bubble interactions when, for example, a bubble breaks at the air-water interface. Another suggests that cells exhibit higher resistanceto shear stress in the presence of the triblocks. Poloxamer 188 has alsobeen reported to facilitate the refolding and to suppress aggregationof a thermally denatured protein [43]. Removal of poloxamer 188during product recovery may compromise the product yield, as wellas 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) bypreferentially locating at an interface, in this way precluding proteinadsorption, and/or (b) by associating with proteins in solution, in thisway stabilizing them against close approach and inhibiting aggregation(Fig. 4). Some surfactants may function according to only one of thesemechanisms, while others may function according to both.

2.3.1. Protein stabilization by surfactant adsorption at interfacesA 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 ofprotein adsorption. Complete hindrance is attributed to the fasterdiffusion of the (generally smaller) surfactant molecules to theinterface, as compared to the much larger protein molecules. Theadsorbed surfactant layer coats the interface, and sterically preventsprotein adsorption. Reduced or increased amounts of adsorption areusually attributed to the formation of surfactant-protein complexeswith reduced or increased surface affinity, respectively. In either case,the behavior of these complexes is considerably different from that ofthe pure protein or surfactant in solution.

An important goal in biotechnology process development andbiopharmaceutical formulation engineering is tominimize the proteinloss that occurs throughout the process by colloidal and interfacialmechanisms, e.g., aggregation and adsorption [45,46]. In order toachieve this, a fundamental understanding of the mechanismsunderlying surfactant effectiveness is necessary. In particular, betterunderstanding 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 alsocommercially available [40].


surfactant-protein complex in modulating interfacial behavior willprovide direction for much-needed process improvements in theproduction and finishing of therapeutic proteins.

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O2.3.1.1. Expectations based on sequential and competitive adsorptionexperiments. The sequential introduction of a surfactant followingprotein adsorption at an interface may result in the removal ofadsorbed 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 ofa stronger surfactant-surface association. The extent of surfactant-mediated removal of adsorbed protein depends on protein, surfactantand surface properties, and also other factors [47]. In general, thedifference in the amount of adsorbed protein eluted by anionic,cationic and nonionic surfactants correlates with the strength ofbinding between the surfactant and the protein in solution [48].Nonionic surfactants, which are known to bind rather weakly toproteins, are least effective in removing adsorbed protein moleculesfrom the interface. In particular, when introduced to an adsorbedprotein layer on a hydrophilic surface, nonionic surfactants generallyhave little effect on the adsorbed amount. In contrast, on hydrophobicsurfaces, nonionic surfactants typically have a substantial effect on theadsorbed protein, presumably because of the difference in surfactantbinding strength at the interface [49].

Joshi andMcGuire [50] have described the interaction of lysozyme,a well-characterized globular protein, with the nonionic surfactantpolysorbate 80 at solid-water interfaces. The concentration of thesurfactant, as well as the method of surfactant and proteinintroduction to the surface (i.e. sequential or combined) was variedin order to elucidate the separate roles of protein, surfactant, and theprotein-surfactant complex in determining adsorption outcomes.They reported a decrease in lysozyme adsorption on hydrophobicsilica upon addition of polysorbate 80, and this reduction in adsorbedprotein increased with the concentration of polysorbate 80 insolution. Sequential adsorption experiments showed that, at suffi-ciently high concentration, polysorbate 80 was able to removeadsorbed lysozyme from a hydrophobic surface. In addition, ifpolysorbate 80 was introduced to the hydrophobic surface prior toaddition of lysozyme, adsorption of the protein was reduced or eveneliminated. On the other hand, polysorbate 80 had no effect on theadsorption of lysozyme onto hydrophilic silica. Finally, sequentialadsorption 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 observeddifferences in protein adsorption were attributed to surface-depen-dent differences in the binding affinity of polysorbate 80 tohydrophobic or hydrophilic surfaces; this work emphasizes theimportance of direct interactions between the surfactant and thesolid surface, relative to surfactant-protein interactions in solution.Accordingly, the rapid diffusion of the small surfactant molecules tothe interface (relative to proteins) is likely to contribute to a reductionin protein adsorption only if the surfactant-surface affinity is suf-ficiently high.



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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 andthus prevent close approach and aggregation.


2.3.1.2. On the stabilization of rFVIII by polysorbate 80 at solid-waterinterfaces. Lysozyme is a much-used “model” protein for the study ofadsorption phenomena in a number of well-controlled circumstances,but results of such work have contributed to forming a foundation forthe greater understanding of the behavior of more complextherapeutic proteins in surfactant-containing formulations. Theadsorption, structural alteration and biological activity of a recombi-nant Factor VIII (rFVIII) was investigated at a hydrophilic andhydrophobic solid-water interface in the presence of polysorbate 80[24]. As in the case of polysorbate 80 and lysozyme, association ofpolysorbate 80 and rFVIII in solution was observed to be entirelyineffective in reducing rFVIII adsorption, indicating that the surfactantprevents adsorption by coating the interface, not the individualprotein molecules. Moreover, observations were attributed to the

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Fig. 5. Schematic illustration of surface tension depression associated with five regimes of pro


PRO

OFhigh 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 withsurfactant addition under appropriate circumstances, or in generalthrough the application of so-called “nonfouling” coatings, such asthose exhibiting pendant PEO chains [52–55]. The pendant polymerchains resist protein adsorption by several mechanisms, primarilysteric repulsion [56]. It is thus reasonable that steric repulsion is arequirement for eliminating protein adsorption, and explains thenonfouling effect of such coatings. In the context of the worksummarized in relation to polysorbate 80, steric repulsion adequately

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tein-surfactant and surfactant-interface interactions. Redrawn with permission from [58].



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Fig. 6. Schematic of differences in kinetics and extent of surface tension depression bysurfactant alone (S) or surfactant with protein (S+P), at different surfactantconcentrations ([S]) for a constant protein concentration. In general, surface tensiondepression increases faster at a given [S] in the presence of protein. Steady-state surfacetensions corresponding to S and S+P converge at sufficiently high [S].


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explains the protein-repellent effect of added surfactants in thepresence of surfaces for which surfactant-surface binding is strong,and the absence of any significant effect of added surfactant withsurfaces for which the surfactant-surface binding is weak. It followsthat steric repulsion is a requirement for the surfactant-mediatedprevention of protein aggregation.

2.4. Protein stabilization by surfactant-protein association

Protein stabilization by association of surfactants requires onlysufficiently strong surfactant-protein interaction, and would beeffective in reducing protein adsorption, regardless of the strengthof surfactant-surface binding. It is instructive to consider thismechanism of protein stabilization, with reference to results ofsurfactant action recorded at air-water interfaces in the presence andabsence of proteins.

2.4.1. A simple view of surfactant-protein mixtures at the air-waterinterface

The molecular dynamics contributing to changes in air-waterinterfacial tension for protein-surfactant mixtures are complex, butoffer an insight into the mechanisms of surfactant interactions withinterfaces and proteins. In ideal circumstances (i.e., for random chainprotein molecules and small, ionic surfactants) the followingequilibrium behavior is expected with increasing surfactant concen-tration (Fig. 5) [57,58]:

Region 1. At very low surfactant concentrations, the steady-stateinterfacial tension is the same as it would be for a pure proteinsolution. The relatively few surfactant molecules have little or noeffect on surface tension.

Region 2. As surfactant concentration increases, the interfacialtension decreases, due to surfactant occupation of “empty sites” atthe air-water interface, as well as the formation of surface-activeprotein-surfactant complexes.

Region 3. At higher surfactant concentrations, the interfacial tensionis expected to plateau, presumably because it is energeticallyfavorable for surfactant to bind to protein at these concentrations(in this range, the CMC recorded for the surfactant in protein-freebuffer may be exceeded).

Region 4. Equilibrium interfacial tension decreases again withincreasing 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 effecton interfacial tension, and a second plateau is reached. In this regime,the CMC, which is specific to the protein concentration used in theexperiment, has been reached, and no further surfactants can adsorbto the air-water interface [59].

This description relates to an idealized protein-surfactant mixtureat equilibrium, and is a useful reference for interpreting observations ofsystems of greater complexity. However, for theoretical and practicalreasons, measurements of the true interfacial equilibrium are usuallynot possible for real protein-surfactant mixtures. This is due mainlyto the inherent irreversibility of protein adsorption, as well asuncertainties 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 anumber of systems. Comparison of the steady-state surface tensionrecorded for surfactant-protein mixtures with that of surfactant alone(at similar concentrations) can be used to reveal whether proteinadsorption is evident, or if the steady-state interfacial behavior is


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governed entirely by surfactant. In particular, if no appreciabledifference is recorded between the steady-state value of interfacialtension demonstrated by protein-surfactant mixtures and by thesurfactant alone at a similar concentration, one may tentativelyconclude that only the surfactant undergoes appreciable adsorption atthe interface (Fig. 6). For example, air-water interfacial tensiometryexperiments were performed with mixtures of rFVIII and polysorbate80. The measured steady-state interfacial tensions, with and withoutthe 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 pulmonarysurfactant

Schram and Hall [60] determined the influence of the twohydrophobic proteins, SP-B and SP-C, on the thermodynamic barriersthat limit the adsorption of pulmonary surfactant vesicles to the air–water interface in the lung. Vesicle adsorption, in this case, ischaracterized by separation of the surfactant acyl chains, followedby fusion of the bilayer vesicle with the interface to form a monolayer(Fig. 7).

For this purpose they measured the kinetics of adsorption (basedon interfacial tensiometry) for intact calf lung surfactant extract, andcompared themwith adsorption of an extract containing the completeset of surfactant lipids, but depleted of the SP-B and SP-C proteins. Thesurfactant 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 wasaccurately reflected in the surface tension kinetic results recorded bySchram and Hall. They interpreted their kinetic results for intact andprotein-free surfactant adsorption with reference to a mechanism forvesicle adsorption. They postulated that vesicle adsorption and fusionis governed by the formation of a rate-limiting structural intermediatebetween the free and adsorbed forms (Fig. 7).

In particular they measured the rate constant (km) characterizingthe slope of the surface tension–time isotherm during the initialdecrease in surface tension, at each of a series of differenttemperatures and concentrations, according to:

rate = km⋅ cn; ð1Þ

where n, the order of the reaction, was obtained from measurementsof the initial adsorption rate at each concentration, c, in the bulkphase.



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The activation energies, Ea, for adsorption were then derived fromthe slopes of plots of the experimental ln km vs. 1/T data, according tothe Arrhenius equation:

ln km = − EaR ⋅

1T

� �+ ln A; ð2Þ

where R is the gas constant, T is temperature, and A is the Arrheniuspre-exponential factor. They invoked transition-state theory toconsider the expected effect of temperature, in terms of anequilibrium between the “reactants” (i.e., the vesicles and unoccupiedair-water interface) and an activated complex. From this model, therate constant can be described in thermodynamic terms:

km =kbTh

� �e−ΔG=RT

; ð3Þ

where kb andh are Boltzmann's andPlanck's constants, respectively, andΔG is the Gibbs free energy of transition. Thus, since ΔG=ΔΗ−ΤΔS,

lnkmT

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The slope and intercept of plots of ln km/T vs. 1/T therefore providequantitative estimates of the enthalpy (ΔH) and entropy (ΔS) of thetransition.

The transition of a bilayer to form an interfacial monolayer requiresa transient exposure of the hydrophobic tails of the surfactant lipids tothe aqueous environment (Fig. 7), and consequently has an unfavorableentropy of transition. Schram and Hall's analysis, however, showedthat the surfactant proteins did not affect the entropy of transition;rather, the essential effect of the proteins was to minimize anunfavorable enthalpy barrier to formation of the structural intermedi-ate. This enthalpic cost was attributed to the dissociation of thesurfactant acyl chains during the separation of the leaves of the bilayers.

An interesting observation characteristic of surface tensiondepression by surfactant-protein mixtures is that the kinetics ofsurface tension depression in such mixtures tend to be uniformlygreater than that recorded for surfactant alone at the sameconcentration, regardless of whether the final steady-state surfacetension is similar in each case (Fig. 6). This kind of “synergistic” effectis 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 greaterfor polysorbate 80-rFVIII mixtures than for polysorbate acting alone,at all polysorbate concentrations studied in that work (8 to 108 ppm).

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Fig. 7. Schematic of the rate-limiting intermediate state in the transition from aphospholipid bilayer to an interfacial monolayer. Proteins located within the bilayerwere found to reduce the enthalpic barrier of the intermediate state. Adapted from [60]with permission from Elsevier.


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The reasons for this have not been articulated in any quantitativefashion, but might be explained in part using an approach to theproblem similar to that outlined by Schram and Hall [60]. Thisapproach might also provide direction for surfactant selection (orsurfactant design) to more effectively manage issues surroundingaggregation and adsorption loss.

3.2. Surfactant-protein association at surfactant concentrations abovethe CMC

Consider the case of surfactant adsorption in the presence ofprotein, at surfactant concentrations above the CMC (or otherwiseconsistent with the presence of associated unimers, if the surfactantsystem is not governed by any obvious CMC). The hydrophobicinteractions that maintain the structure of a micelle or othersurfactant aggregate are based on entropy. Thus a thermodynamicanalysis similar to that used by Schram and Hall [60] can be applied tothe notion of protein-mediated acceleration of surfactant adsorptionintroduced in Section 3.1. We suggest that proteinsmay accelerate theadsorption of surfactants by reducing the major entropic barrier facedby the surfactant in moving from the aggregate to interface, and/or byreducing the enthalpy of activation. In the latter case, the proteinsmight destabilize the surfactant self-association, or they couldproduce a “catalytic” reduction in the enthalpy of some structuralintermediate between unbound surfactant aggregates and adsorbedsurfactant unimers (Fig. 8). In either case, the likely source for achange in enthalpy would be an alteration of the van der Waalsinteractions among the regions on the surfactant moleculesmediatingtheir self-association. Separation of surfactant monomers fromaggregates and their location at the air-water interface would disruptvan 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 andthe adsorption kinetics exhibited by poloxamers, for example,strongly depends on their solution concentration during adsorption[62,63]. At low concentrations, the triblocks exist as individualmolecules (unimers), and their adsorption may not uniformly coverthe entire available surface. At high concentrations, however,adsorption is dominated by micelles or other aggregates, and thePEO chains may inhibit the association between the hydrophobiccenter blocks and the surface. Adsorption of poloxamers is slow incomparison 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 ofselected proteins and, for example, poloxamer 188. We maytentatively 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 belowthe CMC

There is convincing evidence that poloxamers interact with theplasma membranes of cells. More hydrophobic poloxamers generallyshow greater tendencies to incorporate into the cell plasmamembrane [63]. Gigout et al. [64] examined the incorporation ofpoloxamer 188 into the cell plasma membrane and its subsequentuptake by chondrocytes and CHO cells. They were able to conclusivelydemonstrate that the triblocks did in fact enter the cells, and possiblyaccumulate in the endocytic pathway.

While poloxamer 188 shows a high affinity for entropically-drivenassociations with cell membranes, it is not expected to form micellesin water except at very high concentrations [38,40,41,65]. It ispossible that the observed high affinity for cell surfaces can beexplained by the significantly decreased solubility of the surfactant inthe presence of salts. Patel et al. studied the micellization of a very



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hydrophilic poloxamer (80% PEO) in various sodium salt solutions.While the triblock did not form micelles in pure water at ambienttemperatures, it exhibited aggregation and micellization in saltsolutions. The cloud point and critical micelle temperature (CMT)decreased substantially in the presence of salts. They found that theaverage micelle size increased with salt concentration, and therelative effect of the different anions to enhance micellizationfollowed the Hofmeister series (i.e. PO4

3–NSO42–NF–NCl–NBr–), con-

sistent with salting out of the poloxamer [66].Clouding is a thermodynamic phase transition that is characteristic

of molecules containing PEO or other polyethers, and is commonlyassociated with triblocks and other nonionic surfactants in water. It isdue in part to changes in hydrogen bonding and increasinglyattractive interactions between the PEO chains as the temperatureincreases, making the water a “less good” solvent [67]. The influenceof salts can be explained in a similar fashion, being primarily causedby the existence of a salt-deficient zone surrounding the PEO chains:small ions with little polarizability would be repelled by the poorlypolarizable PEO chain. This salt-deficient zone gives rise to anattractive component in the interaction between PEO segments. Aspolymer segments approach each other, their salt depletion zonesoverlap and the surrounding watermolecules are liberated to the bulksolution. The excluded water molecules have a lower chemicalpotential in the bulk, a thermodynamically favored state [38].

Cell surfaces (as well as the “surfaces” of proteins) are charged and“separated” from the bulk solution by a surrounding ion-enrichedelectrical double layer [68,69]. It is reasonable to anticipate a markeddecrease in solubility of a poloxamer at the vicinity of such a colloidalsurface, owing to the higher salt concentration at the interface than inthe bulk. It is thus tempting to hypothesize that a surfactant unimerthat approaches a cell or protein “surface”would find itself in a regionof higher ionic strength, and be driven to associate with the surface bya “salting out”mechanism. Thus, surfactants could effectively stabilizea protein by association with the surface, forming a surfactant-proteincomplex that guards against close approach and subsequent aggre-gation with other proteins. Conversely, the surface at an air-waterinterface is depleted of salts relative to the bulk solution [70], andsurfactant unimers at the surface would encounter a “more watery”micro-environment, and would thus not adsorb with high affinity tothe interface. The effects of these microscopic changes in ionicstrength are consistent with the accelerated surface tension depres-sion observed for surfactant-proteinmixtures (relative to protein-freesurfactant), and also with protein stabilization in the vicinity of theair-water interface, even when the interface is only partially occupied


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Rby surfactant. Further research into these mechanisms is clearlywarranted.

4. Conclusions

Surfactants stabilize proteins by their preferential location at aninterface and/or their association with protein in solution. In thecommon case of surfactant-protein interaction governed by hydro-phobic association, the molecular origins of surfactant-mediatedstabilization of protein can be inferred, in part, from quantitativemodels. Insights can be gained by comparison of thermodynamicbarriers that limit surfactant adsorption, from surfactant-proteinmixtures and protein-free surfactant solutions, to the air–waterinterface. These barriers are defined by the enthalpy and entropy oftransition from free (whether unimeric or aggregated) surfactantmolecules to adsorbed surfactants, via formation of a rate-limitingstructural intermediate. This is easily done, for example, throughanalysis of surface tension kinetic data recorded at differenttemperatures. Concentration effects on the aggregation state of asurfactant, effects of salt on micellization, and the thermodynamicbarriers to its adsorption are particularly important factors indetermining the effectiveness of a given surfactant at stabilizing aprotein. A fundamental understanding of the mechanisms ofaggregation and how surfactants interact with interfaces and proteins(particularly the preferential location of a surfactant at an interface, orits association with protein in solution), provides guidance inselecting surfactants and excipients to reduce protein losses in agiven application.

Acknowledgments

The authors are grateful to David Brems and Sekhar Kanapuram atAmgen for helpful discussions during the preparation of thismanuscript.

References

[1] I.S. Johnson, Human insulin from recombinant DNA technology, Science 219(1983) 632–637.

[2] J.S. Bee, T.W. Randolph, J.F. Carpenter, S.M. Bishop, M.N. Dimitrova, Effects ofsurfaces and leachables on the stability of biopharmaceuticals. J. Pharm. Sci. 100(2011) in press DOI:10.1002/jps.22597.

[3] J. Carpenter, B. Cherney, A. Lubinecki, S. Ma, E. Marszal, A. Mire-Sluis, T. Nikolai, J.Novak, J. Ragheb, J. Simak, Meeting report on protein particles and immunoge-nicity of therapeutic proteins: filling in the gaps in risk evaluation and mitigation,Biologicals 38 (2010) 602–611.



T

891892893894895896897898899900901902903904905906907908909910911912913914915916917918919920921922923924925926927928929930931932933934935936937938939940941942943944945946947948949950951952953954955956957958959960961962963964965966967968969970971972973974975976

977978979980981982983984985986987988989990991992993994995996997998999100010011002100310041005100610071008100910101011101210131014101510161017101810191020102110221023102410251026102710281029103010311032103310341035103610371038103910401041104210431044104510461047104810491050105110521053105410551056105710581059106010611062


UNCO

RREC

[4] W. Wang, S. Nema, D. Teagarden, Protein aggregation – pathways and influencingfactors, Int. J. Pharm. 390 (2010) 89–99.

[5] J.S. Philo, T. Arakawa, Mechanisms of protein aggregation, Curr. Pharm.Biotechnol. 10 (2009) 348–351.

[6] E.Y. Chi, S. Krishnan, T.W. Randolph, J.F. Carpenter, Physical stability of proteins inaqueous solution: Mechanism and driving forces in nonnative protein aggrega-tion, Pharm. Res. 20 (2003) 1325–1336.

[7] A.A. Raibekas, E.J. Bures, C.C. Siska, T. Kohno, R.F. Latypov, B.A. Kerwin, Anionbinding and controlled aggregation of human interleukin-1 receptor antagonist,Biochemistry 44 (2005) 9871–9879.

[8] R.S. Rajan, T. Li, M. Aras, C. Sloey, W. Sutherland, H. Arai, R. Briddell, O. Kinstler,A.M. Lueras, Y. Zhang, H. Yeghnazar, M. Treuheit, D.N. Brems, Modulation ofprotein aggregation by polyethylene glycol conjugation: GCSF as a case study,Protein Sci. 15 (2006) 1063–1075.

[9] T.A. Horbett, Protein adsorption on biomaterials, in: S.L. Cooper, N.A. Peppas, A.S.Hoffman, B.D. Ratner (Eds.), Biomaterials: Interfacial Phenomena and Applica-tions, American Chemical Society, Washington D.C, 1982, pp. 233–244.

[10] R. Nezlin, Interactions between immunoglobulin G molecules, Immunol. Lett. 132(2010) 1–5.

[11] K. Giger, R.P. Vanam, E. Seyrek, P.L. Dubin, Suppression of insulin aggregation byheparin, Biomacromolecules 9 (2008) 2338–2344.

[12] D.S. Katayama, R. Nayar, D.K. Chou, J.J. Valente, J. Cooper, C.S. Henry, D.G. VanderVelde, L. Villarete, C.P. Liu, M.C. Manning, Effect of buffer species on the thermallyinduced aggregation of interferon-tau, J. Pharm. Sci. 95 (2006) 1212–1226.

[13] S. Krishnan, E.Y. Chi, J.N. Webb, B.S. Chang, D. Shan, M. Goldenberg, M.C. Manning,T.W. Randolph, J.F. Carpenter, Aggregation of granulocyte colony stimulatingfactor under physiological conditions: characterization and thermodynamicinhibition, Biochemistry 41 (2002) 6422–6431.

[14] S.N. Timasheff, Control of protein stability and reactions by weakly interactingcosolvents: the simplicity of the complicated, Adv. Protein Chem. 51 (1998) 355–432.

[15] T. Arakawa, Y. Kita, J.F. Carpenter, Protein–solvent interactions in pharmaceuticalformulations, Pharm. Res. 8 (1991) 285–291.

[16] N.A. Ciaccio, J.S. Laurence, Effects of disulfide bond formation and protein helicityon the aggregation of activating transcription factor 5, Mol. Pharm. 6 (2009)1205–1215.

[17] D. Liu, D. Ren, H. Huang, J. Dankberg, R. Rosenfeld, M.J. Cocco, L. Li, D.N. Brems, R.L.Remmele Jr., Structure and stability changes of human IgG1 Fc as a consequence ofmethionine oxidation, Biochemistry 47 (2008) 5088–5100.

[18] M.R. Nilsson, M. Driscoll, D.P. Raleigh, Low levels of asparagine deamidation canhave a dramatic effect on aggregation of amyloidogenic peptides: Implications forthe study of amyloid formation, Protein Sci. 11 (2002) 342–349.

[19] J.S. Bee, S.A. Nelson, E. Freund, J.F. Carpenter, T.W. Randolph, Precipitation of amonoclonal antibody by soluble tungsten, J. Pharm. Sci. 98 (2009) 3290–3301.

[20] R. Thirumangalathu, S. Krishnan, M.S. Ricci, D.N. Brems, T.W. Randolph, J.F.Carpenter, Silicone oil- and agitation-induced aggregation of a monoclonalantibody in aqueous solution, J. Pharm. Sci. 98 (2009) 3167–3181.

[21] D.K. Chou, R. Krishnamurthy, T.W. Randolph, J.F. Carpenter, M.C. Manning, Effectsof Tween 20® and Tween 80® on the stability of albutropin during agitation,J. Pharm. Sci. 94 (2005) 1368–1381.

[22] O. Joshi, L. Chu, J. McGuire, D.Q. Wang, Adsorption and function of recombinantFactor VIII at the air-water interface in the presence of Tween 80®, J. Pharm. Sci.98 (2009) 3099–3107.

[23] L.A. Kueltzo, W. Wang, T.W. Randolph, J.F. Carpenter, Effects of solutionconditions, processing parameters, and container materials on aggregation of amonoclonal antibody during freeze-thawing, J. Pharm. Sci. 97 (2008) 1801–1812.

[24] O. Joshi, J. McGuire, D.Q. Wang, Adsorption and function of recombinant factor VIIIat solid–water interfaces in the presence of Tween-80®, J. Pharm. Sci. 97 (2008)4741–4755.

[25] T.W. Randolph, L.S. Jones, Surfactant-protein interactions, in: J.F. Carpenter, M.C.Manning (Eds.), Rational Design of Stable Protein Formulations: Theory andPractice, Kluwer Academic, New York, 2002, pp. 159–175.

[26] I.W. Hamley, Block Copolymers, Solution: Fundamentals And Applications, Wiley,Chichester, England, 2005, pp. 18–20.

[27] P. Garidel, C. Hoffmann, A. Blume, A thermodynamic analysis of the bindinginteraction between polysorbate 20 and 80 with human serum albumins andimmunoglobulins: A contribution to understand colloidal protein stabilization,Biophys. Chem. 143 (2009) 70–78.

[28] Y.R. Gokarn, A. Kosky, E. Kras, A. McAuley, R.L. Remmele Jr., Excipients for proteindrugs, in: A. Katdare, M.V. Chaubal (Eds.), Excipient Development For Pharma-ceutical, Biotechnology, and Drug Delivery Systems, Informa Healthcare,New York, 2006, pp. 291–331.

[29] S. Deechongkit, J. Wen, L.O. Narhi, Y. Jiang, S.S. Park, J. Kim, B.A. Kerwin, Physicaland biophysical effects of polysorbate 20 and 80 on darbepoetin alfa, J. Pharm. Sci.98 (2009) 3200–3217.

[30] L. Kreilgaard, L.S. Jones, T.W. Randolph, S. Frokjaer, J.M. Flink, M.C. Manning, J.F.Carpenter, Effect of Tween 20® on freeze-thawing- and agitation-inducedaggregation of recombinant human factor XIII, J. Pharm. Sci. 87 (1998) 1597–1603.

[31] J.F. Carpenter, M.J. Pikal, B.S. Chang, T.W. Randolph, Rational design of stablelyophilized protein formulations: Some practical advice, Pharm. Res. 14 (1997)969–975.

[32] B.A. Kerwin, M.C. Heller, S.H. Levin, T.W. Randolph, Effects of Tween 80® andsucrose on acute short-term stability and long-term storage at −20 °C of arecombinant hemoglobin, J. Pharm. Sci. 87 (1998) 1062–1068.

[33] H.C. Mahler, R. Muller, W. Friess, A. Delille, S. Matheus, Induction and analysis ofaggregates in a liquid IgG1-antibody formulation, Eur. J. Pharm. Biopharm. 59(2005) 407–417.


ED P

RO

OF

[34] S. Kiese, A. Papppenberger, W. Friess, H.-C. Mahler, Shaken, not stirred:Mechanical stress testing of an IgG1 antibody, J. Pharm. Sci. 97 (2008) 4347–4366.

[35] R.S. Kishore, A. Pappenberger, I.B. Dauphin, A. Ross, B. Buergi, A. Staempfli, H.C.Mahler, Degradation of polysorbates 20 and 80: Studies on thermal autoxidationand hydrolysis, J. Pharm. Sci. 100 (2010) 721–731.

[36] W. Wang, Y.J. Wang, D.Q. Wang, Dual effects of Tween 80® on protein stability,Int. J. Pharm. 347 (2008) 31–38.

[37] A.V. Kabanov, P. Lemieux, S. Vinogradov, V. Alakhov, Pluronic® block copolymers:Novel functional molecules for gene therapy, Adv. Drug Deliv. Rev. 54 (2002)223–233.

[38] P. Bahadur, P. Li, M. Almgren, W. Brown, Effect of potassium fluoride on themicellar behavior of Pluronic® F-68 in aqueous solution, Langmuir 8 (1992)1903–1907.

[39] P. Alexandridis, J.F. Holzwarth, T.A. Hatton, Micellization of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymers in aqueoussolutions: Thermodynamics of copolymer association, Macromolecules 27 (1994)2414–2425.

[40] H.-W. Tsui, Y.-H. Hsu, J.-H. Wang, L.-J. Chen, Novel behavior of heat ofmicellization of Pluronics® F-68 and F-88 in aqueous solutions, Langmuir 24(2008) 13858–13862.

[41] Y. Li, M. Bao, Z. Wang, H. Zhang, G. Xu, Aggregation behavior and complexstructure between triblock copolymer and anionic surfactants, J. Mol. Struct. 985(2011) 391–396.

[42] T. Tharmalingam, H. Ghebeh, T. Wuerz, M. Butler, Pluronic® enhances therobustness and reduces the cell attachment of mammalian cells, Mol. Biotechnol.39 (2008) 167–177.

[43] D. Mustafi, C.M. Smith, M.W. Makinen, R.C. Lee, Multi-block poloxamer surfactantssuppress aggregation of denatured proteins, Biochim. Biophys. Acta 1780 (2008) 7–15.

[44] M. Al-Rubeai, A.N. Emery, S. Chalder, The effect of Pluronic® F-68 on hybridomacells in continuous culture, Appl. Microbiol. Biotechnol. 37 (1992) 44–45.

[45] E.Y. Chi, J. Weickmann, J.F. Carpenter, M.C. Manning, T.W. Randolph, Heteroge-neous nucleation-controlled particulate formation of recombinant humanplatelet-activating factor acetylhydrolase in pharmaceutical formulation, J.Pharm. Sci. 94 (2005) 256–274.

[46] L.S. Jones, A. Kaufmann, C.R. Middaugh, Silicone oil induced aggregation ofproteins, J. Pharm. Sci. 94 (2005) 918–927.

[47] J. McGuire, M.C. Wahlgren, T. Arnebrant, Structural stability effects on theadsorption and dodecyltrimethylammonium bromide-mediated elutability ofbacteriophage T4 lysozyme at silica surfaces, J. Colloid Interface Sci. 170 (1995)182–192.

[48] T. Arnebrant, M.C. Wahlgren, Protein-surfactant interactions at solid surfaces, in:T.A. Horbett, J.L. Brash (Eds.), Proteins at Interfaces II: Fundamentals andApplications, American Chemical Society, Washington D.C., 1995, pp. 239–254.

[49] H. Elwing, A. Askendal, I. Lundstr, Desorption of fibrinogen and γ-globulin fromsolid surfaces induced by a nonionic detergent, J. Colloid Interface Sci. 128 (1989)296–300.

[50] O. Joshi, J. McGuire, Adsorption behavior of lysozyme and Tween 80® athydrophilic and hydrophobic silica-water interfaces, Appl. Biochem. Biotechnol.152 (2009) 235–248.

[51] Y.C. Tai, J. McGuire, O. Joshi, D.Q. Wang, Solid surface chemical and physical effectson the adsorption of recombinant Factor VIII, Pharm. Dev. Technol. 14 (2009)126–130.

[52] N.P. Desai, J.A. Hubbell, Biological responses to polyethylene oxide modifiedpolyethylene terephthalate surfaces, J. Biomed. Mater. Res. 25 (1991) 829–843.

[53] J.-T. Li, J. Carlsson, S.-C. Huang, K.D. Caldwell, Adsorption of poly(ethylene oxide)-containing block copolymers, in: E. Glass (Ed.), Hydrophilic Polymers: Perfor-mance with Environmental Acceptability, American Chemical Society, Washing-ton D.C., 1996, pp. 61–78.

[54] J.T. Li, K.D. Caldwell, Sedimentation field flow fractionation in the determinationof surface concentration of adsorbed materials, Langmuir 7 (1991) 2034–2039.

[55] M. Paulsson, M. Kober, C. Freij-Larsson, M. Stollenwerk, B. Wesslen, A. Ljungh,Adhesion of Staphylococci to chemically modified and native polymers, and theinfluence of preadsorbed fibronectin, vitronectin and fibrinogen, Biomaterials 14(1993) 845–853.

[56] L.D. Unsworth, H. Sheardown, J.L. Brash, Protein-resistant poly(ethylene oxide)-grafted surfaces: chain density-dependent multiple mechanisms of action,Langmuir 24 (2008) 1924–1929.

[57] E. Dickinson, C.M. Woskett, Competitive adsorption between proteins and small-molecule surfactants in food emulsions, in: R.D. Bee, P. Richmond, J. Mingins(Eds.), Food Colloids, Royal Society of Chemistry, Cambridge, 1989, p. 74.

[58] W.D. Gray, Surfactant effects on adsorption of recombinant Factor VIII (rFVIII) atthe air-water interface. Honors thesis, Oregon State University, Corvallis, Oregon,USA 2009.

[59] M.N. Jones, D. Chapman, Micelles, Monolayers, and Biomembranes, Wiley, NewYork, 1995, pp. 64–101.

[60] V. Schram, S.B. Hall, Thermodynamic effects of the hydrophobic surfactantproteins on the early adsorption of pulmonary surfactant, Biophys. J. 81 (2001)1536–1546.

[61] Y. Bogdanova, V. Dolzhikova, Surface and bulkproperties of aqueous binarymixturesof Pluronic® F68 and low-molecular-weight cationic surfactants: 1. Surface tensionand association in aqueous solutions, Colloid J. 70 (2008) 138–143.

[62] F.A.M. Leermakers, A.P. Gast, Block copolymer adsorption studied by dynamicscanning angle reflectometry, Macromolecules 24 (1991) 718–730.

[63] L.-C. Chang, C.-Y. Lin, M.-W. Kuo, C.-S. Gau, Interactions of Pluronics® withphospholipid monolayers at the air-water interface, J. Colloid Interface Sci. 285(2005) 640–652.



106310641065106610671068106910701071

1072107310741075107610771078

1079


[64] A. Gigout, M.D. Buschmann, M. Jolicoeur, The fate of Pluronic® F-68 inchondrocytes and CHO cells, Biotechnol. Bioeng. 100 (2008) 975–987.

[65] S. Borbely, J.S. Pedersen, Temperature-induced aggregation in aqueous solutionsof Pluronic® F68 triblock copolymer containing small amount of o-xylene, PhysicaB 276–278 (2000) 363–364.

[66] K. Patel, B. Bharatiya, Y. Kadam, P. Bahadur,Micellization and clouding behavior of EO–PO block copolymer in aqueous salt solutions, J. Surfactants Deterg. 13 (2010) 89–95.

[67] H. Schott, S. Han, Effect of inorganic additives on solutions of nonionic surfactantsIII: CMC's and surface properties, J. Pharm. Sci. 65 (1976) 975–978.

UNCO

RRECT


[68] R. Pethig, D.B. Kell, The passive electrical properties of biological systems: theirsignificance in physiology, biophysics and biotechnology, Phys. Med. Biol. 32(1987) 933–970.

[69] V. Raicu, A. Popescu, Integrated Molecular and Cellular Biophysics, Springer,Netherlands, 2008, pp. 78–100.

[70] Y. Levin, A.P. dos Santos, A. Diehl, Ions at the air-water interface: An end to ahundred-year-old mystery? Phys. Rev. Lett. 103 (2009) 257802–257805.

ED P

RO

OF



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