AN ABSTRACT OF THE DISSERTATION OF
Hyo Jin Lee for the degree of Doctor of Philosophy in Chemical Engineering
presented on September 4, 2012
Title: Molecular origins of surfactant- mediated stabilization of protein
Abstract approved:
Joseph McGuire
Nonionic surfactants are commonly used to stabilize proteins during upstream
and downstream processing and drug formulation. Surfactants stabilize the proteins
through two major mechanisms: (i) their preferential location at nearby interfaces, in this
way precluding protein adsorption; and/or (ii) their association with protein into
“complexes” that prevent proteins from interacting with surfaces as well as each other. In
general, both mechanisms must be at play for effective protein stabilization against
aggregation and activity loss, but selection of surfactants for protein stabilization
currently is not made with benefit of any quantitative, predictive information to ensure
that this requirement is met.
In certain circumstances the kinetics of surface tension depression (by surfactant)
in protein-surfactant mixtures has been observed to be greater than that recorded for
surfactant alone at the same concentration. We compared surface tension depression by
poloxamer 188 (Pluronic® F68), polysorbate 80 (PS 80), and polysorbate 20 (PS 20) in
the presence and absence of lysozyme and recombinant protein, at different surfactant
concentrations and temperatures. The kinetic results were interpreted with reference to a
mechanism for surfactant adsorption governed by the formation of a rate-limiting
structural intermediate (i.e., an “activated complex”) comprised of surfactant aggregates
and protein. The presence of lysozyme was seen to increase the rate of surfactant
adsorption in relation to surfactant acting alone at the same concentrations for the
polysorbates while less of an effect was seen for Pluronic® F68. However, the addition
of salt was observed to accelerate the surface tension depression of Pluronic® F68 in the
presence of lysozyme. The addition of a more hydrophobic, surface active protein
(Amgen recombinant protein) in place of lysozyme resulted in greater enhancement of
surfactant adsorption than that recorded in the presence of lysozyme. A simple
thermodynamic analysis indicated the presence of protein caused a reduction in ∆𝑮 for
the surfactant adsorption process, with this reduction deriving entirely from a reduction in
∆𝑯. We suggest that protein accelerates the adsorption of these surfactants by disrupting
their self associations, increasing the concentration of surfactant monomers near the
interface.
Based on these air-water tensiometry results, it is fair to expect that accelerated
surfactant adsorption in the presence of protein (observed with PS 20 and PS 80) will
occur with surfactants that stabilize protein mainly by their own adsorption at interfaces,
and that the absence of accelerated surfactant adsorption (observed with F68) will be
observed with surfactants that form stable surfactant-protein associations. Optical
waveguide lightmode spectroscopy was used to test this expectation. Adsorption kinetics
were recorded for surfactants (PS 20, PS 80, or F68) and protein (lysozyme or Amgen
recombinant protein) at a hydrophilic solid (SiO2-TiO2) surface. Experiments were
performed in sequential and competitive adsorption modes, enabling the adsorption
kinetic patterns to be interpreted in a fashion revealing the dominant mode of surfactantmediated stabilization of protein in each case. Kinetic results confirmed predictions based
on our earlier quantitative analysis of protein effects on surface tension depression by
surfactants. In particular, PS 20 and PS 80 are able to inhibit protein adsorption only by
their preferential location at the interface, and not by formation of less surface active,
protein-surfactant complexes. On the other hand, F68 is able to inhibit protein adsorption
by formation of protein-surfactant complexes, and not by its preferential location at the
interface.
©Copyright by Hyo Jin Lee
September 4, 2012
All rights reserved
Molecular origins of surfactant- mediated stabilization of protein
by
Hyo Jin Lee
A DISSERTATION
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Presented September 4, 2012
Commencement June 2013
Doctor of Philosophy dissertation of Hyo Jin Lee presented on September 4, 2012
APPROVED:
Major Professor, representing Chemical Engineering
Chair of the School of Chemical, Biological and Environmental Engineering
Dean of the Graduate School
I understand that my dissertation will become part of the permanent collection of Oregon
State University libraries. My signature below authorizes release of my dissertation to
any reader upon request.
Hyo Jin Lee, Author
ACKNOWLEDGEMENTS
First and foremost, I would like to express sincere appreciation and gratitude to
my advisor Dr. Joseph McGuire for the continuous support from the beginning
till the end of my Ph.D. study. This work would have never happened if it wasn’t
for his patience, motivation, enthusiasm and immense knowledge.
My sincere thanks also go to Arnold McAuley, Sekhar Kanapuram, and Dave
Brems at Amgen for allowing me to work and study for my Ph.D., providing me
with insightful comments.
I would like to thank my husband-to-be Anthony Kim for being there for me and
being so understanding for all the times I had to leave him by himself when I had
to go to the lab.
Last but not least, I would like to thank my family; my aunts, grandmother,
brother and my parents, Youngwon Lee and Heesook Hwang for giving me
infinite moral support throughout my life.
CONTRIBUTION OF AUTHORS
Dr.Karl Schilke was very much involved in getting all the figures and assisted in editing
Chapter 2.Arnold McAuley was heavily involved in the bi-weekly discussions that
contributed to this dissertation.
TABLE OF CONTENTS
Chapter 1- Introduction ....................................................................................................... 1
Chapter 2- Molecular origins of surfactant-mediated stabilization of protein drugs........... 3
Abstract .................................................................................................................. 4
2.1. Introduction ..................................................................................................... 5
2.2. Managing protein aggregation and adsorption loss ........................................ 6
2.2.1. Mechanisms of aggregation ............................................................ 7
2.2.1.1. Concentration-induced aggregation (Mechanism 1)…...8
2.2.1.2. Aggregation induced by conformational change
(Mechanism 2)……….………………………………………...10
2.2.1.3. Aggregation induced by chemical reaction (Mechanism
3).............................................................................................…11
2.2.1.4. Nucleation-dependent aggregation (Mechanism 4)…...12
2.2.1.5. Surface-induced aggregation (Mechanism 5)………....14
2.2.2. Surfactants used in drug products ................................................ 16
2.2.2.1. Polysorbates .................................................................. 18
2.2.2.2. Poloxamers ................................................................... 19
2.2.3. Surfactant modulation of protein adsorption and aggregation ..... 21
2.2.3.1. Protein stabilization by surfactant adsorption at
interfaces ................................................................................... 21
2.2.3.1.1. Expectations based on sequential and
competitive adsorption experiments ............................. 22
2.2.3.1.2. On the stabilization of rFVIII by polysorbate
80 at solid-water interfaces ........................................... 24
2.2.4. Protein stabilization by surfactant-protein association ................. 25
2.2.4.1. A simple view of surfactant-protein mixtures at the airwater interface ........................................................................... 25
2.3. A testable, thermodynamic argument to guide surfactant selection ............. 28
2.3.1. Insights gained from intact and protein-depleted pulmonary
surfactant ................................................................................................ 28
2.3.2. Surfactant-protein association at surfactant concentrations above
the CMC ………………………………………………………………..32
2.3.3. Surfactant-protein association at surfactant concentrations below
the CMC ................................................................................................. 34
2.4. Conclusions ................................................................................................. 36
2.5. References .................................................................................................... 37
TABLE OF CONTENTS (Continued)
Chapter 3- Protein effects on surfactant adsorption suggest the dominant mode of
surfactant-mediated stabilization of protein ...................................................................... 42
Abstract ................................................................................................................ 43
3.1. Introduction ................................................................................................... 44
3.2. Material and Methods.................................................................................... 47
3.2.1. Protein and surfactants .................................................................. 47
3.2.2.Surface tension measurements ....................................................... 47
3.3. Results ........................................................................................................... 48
3.3.1. Effect of surfactant concentration ................................................. 48
3.3.2. Effect of salt .................................................................................. 51
3.3.3. Effect of temperature and comparison to a simple model for
protein-mediated acceleration of surfactant adsorption .......................... 52
3.3.4. Effect of protein ............................................................................ 58
3.4. Conclusions ................................................................................................... 60
3.5. References ..................................................................................................... 62
Chapter 4- Dominant mode of surfactant-mediated protein stabilization of protein at solid
surfaces .............................................................................................................................. 64
Abstract ................................................................................................................ 65
4.1. Introduction ................................................................................................... 66
4.2. Material and Methods.................................................................................... 67
4.2.1. Materials and sample preparation.................................................. 67
4.2.2. Evaluation of adsorption kinetics .................................................. 68
4.2.2.1. Surfactant+ protein co-adsorption ................................. 69
4.2.2.2. Surfactant+protein sequential adsorption ...................... 69
4.2.2.3. Surfactant+protein co-adsorption pre-coated with
surfactant .................................................................................... 70
4.3. Results and Discussion .................................................................................. 70
4.3.1. Surfactant+lysozyme co-adsorption .............................................. 70
4.3.2. Pluronic® F68+lysozyme sequential adsorption .......................... 73
4.3.3. Polysorbates+lysozyme co-adsorption pre-coated with
polysorbates............................................................................................. 74
4.3.4. Surfactants+ recombinant protein co-adsorption........................... 75
4.3.5. Polysorbates+recombinant protein co-adsorption pre-coated with
polysorbates ............................................................................................ 78
TABLE OF CONTENTS (Continued)
4.3.6. Effect of salt on Pluronic® F68+recombinant protein coadsorption and sequential adsorption ..................................................... 79
4.4. Conclusions ................................................................................................... 83
4.5. References ..................................................................................................... 84
Chapter 5-General conclusions ........................................................................................... 85
Bibliography ....................................................................................................................... 87
LIST OF FIGURES
Figure 2.1: Schematic illustration of common mechanisms of aggregation. ..................... 9
Figure 2.2: Chemical structure of polysorbate surfactants. .............................................. 18
Figure 2.3: Chemical structure of the PEO-PPO-PEO triblock copolymer Poloxamer 188.
.......................................................................................................................................... 21
Figure 2.4: 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. ............................................. 21
Figure 2.5: Schematic illustration of surface tension depression associated with five
regimes of protein-surfactant and surfactant-interface interactions. ................................ 27
Figure 2.6: 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]) or a constant protein concentration. teady-state surface tensions
corresponding to S and S + P converge at sufficiently high [S]. ...................................... 28
Figure 2.7: Schematic of the rate-limiting intermediate state in the transition from a
phospholipid bilayer to an interfacial monolayer. ............................................................ 29
Figure 2.8: 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). ........... 33
Figure 3.1: In the absence of protein (top), surfactant monomers must dissociate and
migrate through the liquid in order to adsorb. The presence of protein may facilitate
aggregate disruption, leading either to an increased concentration of surfactant monomers
thus enhancing adsorption (middle), or to the formation of stable, surfactant-protein
complexes having little or no effect on surfactant adsorption rate (bottom). ................... 46
Figure 3.2: Adsorption isotherm (surface tension vs. time) of PS 80 and lysozyme
mixture at 25 °C in 1X PBS. ............................................................................................ 49
Figure 3.3: Adsorption isotherm (surface tension vs. time) of PS 20 and lysozyme
mixture at 25 °C in 1X PBS. ............................................................................................ 49
Figure 3.4: Adsorption isotherm (surface tension vs. time) of Pluronic® F68 and
lysozyme mixture at 25 °C in 1X PBS. ............................................................................ 50
Figure 3.5: Effect of salt on the adsorption isotherm (surface tension vs. time) of 10 ppm
Pluronic® F68 and lysozyme mixture .............................................................................. 52
LIST OF FIGURES (Continued)
Figure 3.6: Effect of salt on the adsorption isotherm (surface tension vs. time) of
surfactants (50 ppm Pluronic® F68, PS 80 and PS 20) and lysozyme mixture ............... 52
Figure 3.7: Adsorption isotherm (surface tension vs. time) of 5 ppm PS 80 and lysozyme
mixture at 5 °C, 25 °C, and 40 °C in 1X PBS. ................................................................. 53
Figure 3.8: Adsorption isotherm (surface tension vs. time) of 5 ppm PS 20 and lysozyme
mixture at 5 °C, 25 °C, and 40 °C in 1X PBS. ................................................................. 54
Figure 3.9: Adsorption isotherm (surface tension vs. time) of 5 ppm Pluronic ® F68 and
lysozyme mixture at 5 °C, 25 °C, and 40 °C in 1X PBS. ................................................. 54
Figure 3.10: Arrhenius plot of adsorption during initial surface tension depression. ...... 56
Figure 3.11: Determination of thermodynamic components of the activation barrier.. ... 57
Figure 3.12: Adsorption isotherm (surface tension vs. time) of PS80 and recombinant
protein mixture at 25 °C in 10 mM sodium acetate, 5% sorbitol, pH 3.5 ........................ 59
Figure 3.13: Adsorption isotherm (surface tension vs. time) of PS 20 and recombinant
protein mixture at 25 °C in 10 mM sodium acetate, 5% sorbitol, pH 3.5 ........................ 60
Figure 3.14. Adsorption isotherm (surface tension vs. time) of Pluronic® F68 and
recombinant protein mixture at 25 °C in 10 mM sodium acetate, 5% sorbitol, pH 3.5 ... 60
Figure 4.1: Adsorption kinetics of 90 and 900 ppm PS 80, 90 and 900 ppm PS 20, and
600 and 6000 ppm Pluronic® F68 in PBS ....................................................................... 71
Figure 4.2: Adsorption kinetics of lysozyme in PBS in the presence of 0 ppm, 900 ppm
PS 80 and 900 ppm PS 20 ................................................................................................ 72
Figure 4.3: Adsorption kinetics of lysozyme in PBS in the presence of 0 ppm, 600 ppm,
and 6000 ppm Pluronic® F68 .......................................................................................... 73
Figure 4.4: Adsorption kinetics of 600 ppm Pluronic® F68 followed by buffer elution
and the introduction of lysozyme in PBS. ........................................................................ 74
Figure 4.5: 90 ppm polysorbates adsorption followed by 90 ppm polysorbates + lysozyme
co-adsorption in PBS ........................................................................................................ 75
Figure 4.6: Adsorption kinetics of 90 and 900 ppm PS 80, 90 and 900 ppm PS 20, and
600 and 6000ppm Pluronic® F68 in 10 mM sodium acetate, 5 % sorbitol, at pH 4.0..... 77
LIST OF FIGURES (Continued)
Figure 4.7: Adsorption kinetics of recombinant protein in 10 mM sodium acetate, 5%
sorbitol, pH 4.0 in in the presence of 0 ppm, 900 ppm PS 80, 900 ppm PS 20, and
6000ppm Pluronic® F68 ...............................................................................................…78
Figure 4.8: 90 ppm polysorbates adsorption followed by 90 ppm polysorbates +
recombinant protein co-adsorption in 10 mM sodium acetate, 5 % sorbitol, pH 4.0 ....... 79
Figure 4.9: Effect of salt concentration on the adsorption of recombinant protein in 10
mM sodium acetate, 5 % sorbitol, at pH 4.0 .................................................................... 81
Figure 4.10: Adsorption kinetics of recombinant protein in 10 mM sodium acetate, 5 %
sorbitol, 250 mM sodium chloride, at pH 4.0 in the presence of 0 ppm, 600ppm, and
6000ppm Pluronic® F68 .................................................................................................. 82
Figure 4.11: Adsorption kinetics of 600ppm Pluronic® F68 followed by buffer elution
and the introduction of recombinant protein in 10 mM sodium acetate, 5 % sorbitol, 250
mM sodium chloride, at pH 4.0. ....................................................................................... 83
MOLECULAR ORIGINS OF SURFACTANT-MEDIATED STABILIZATION OF
PROTEIN
CHAPTER 1
INTRODUCTION
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. Several mitigation strategies are used in the
biotechnology industries to stabilize therapeutic proteins, but the addition of surfactants
appears to be a general approach. Nonionic surfactants are used to protect and stabilize
proteins against adsorption and surface-induced activity loss as well as aggregation in
solution, either by binding to the proteins and preventing protein-protein associations, or
by saturating the interface and thus minimizing adsorption and subsequent
conformational changes. The following chapters encapsulate the work done to better
understand the roles of surfactants and surfactant-protein complexes in modulating
interfacial behavior and aggregation.
Chapter 2 summarizes the roles of surfactants, proteins, and surfactantprotein complexes in modulating interfacial behavior and aggregation. These events
depend on surfactant properties that may be quantified using a thermodynamic model, to
provide physical/chemical direction for surfactant selection or design, and to effectively
reduce aggregation and adsorption loss. A fundamental understanding of the mechanisms
of aggregation and how surfactants interact with interfaces and proteins (particularly the
preferential location of a surfactant at an interface, or its association with protein in
2
solution), provides guidance in selecting surfactants and excipients to reduce protein
losses in a given application.
Chapter 3 is a study of surface tension depression by poloxamer 188
(Pluronic® F68), polysorbate 80 (PS80), and polysorbate 20 (PS20) in the presence and
absence of lysozyme and recombinant protein, at different surfactant concentrations and
temperatures. The kinetic results are interpreted with reference to a mechanism for
surfactant adsorption governed by the formation of a rate-limiting structural intermediate
(i.e., an “activated complex”) between surfactant aggregates and protein.
Chapter 4 explores adsorption of lysozyme and recombinant protein at
solid/liquid interfaces in relation to poloxamer 188 (Pluronic® F68), polysorbate 80
(PS80), and polysorbate 20 (PS20) at different surfactant concentrations. Adsorption
kinetics were recorded for surfactants and protein in sequential and competitive
adsorption modes, enabling the adsorption kinetic patterns to be interpreted in a fashion
revealing the dominant mode of surfactant-mediated stabilization of protein in each case.
These results confirmed predictions based on our earlier quantitative analysis of protein
effects on surface tension depression by surfactants.
Finally, major conclusions made in this overall study are summarized in
Chapter 5.
3
MOLECULAR ORIGINS OF SURFACTANT-MEDIATED STABILIZATION OF
PROTEIN DRUGS
Hyo Jin Lee,a,b Arnold McAuley,b Karl F. Schilkea and Joseph McGuirea,*
a
School of Chemical, Biological and Environmental Engineering, Oregon State University,
Corvallis, OR 97331
b
*
Proces and Product Development Department Amgen Inc., Thousand Oaks, CA 91320
Corresponding Author:
Joseph McGuire
School of Chemical, Biological and Environmental Engineering
Oregon State University
103 Gleeson Hall
Corvallis, OR 97331-2702
Tel:
541-737-6306
Fax:
541-737-4600
Email: mcguirej@engr.orst.edu
4
CHAPTER 2
MOLECULAR ORIGINS OF SURFACTANT-MEDIATED STABILIZATION OF
PROTEIN DRUGS
Abstract
Loss of activity through aggregation and surface-induced
denaturation is a significant problem in the production, formulation and
administration of therapeutic proteins. Surfactants are commonly used in
upstream and downstream processing and drug formulation. However, the
effectiveness of a surfactant 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
associations. Minimizing protein loss from colloidal and interfacial interaction
requires a fundamental understanding of the molecular factors underlying
surfactant effectiveness and mechanism. These concepts provide direction for
improvements in the manufacture and finishing of therapeutic proteins. We
summarize the roles of surfactants, proteins, and surfactant-protein complexes in
modulating interfacial behavior and aggregation. These events depend on
surfactant properties that may be quantified using a thermodynamic model, to
provide physical/chemical direction for surfactant selection or design, and to
effectively reduce aggregation and adsorption loss.
5
2.1. 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 advanced 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.
Therapeutic 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 [2].
Proteins can often be stabilized against adsorption loss through the use of
properly-chosen surfactants. Preferential location of surfactant molecules at interfaces,
6
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 stabilization of the proteins against aggregation and adsorption loss.
2.2. 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, aggregation can often be inhibited by
modifying the molecular properties or by changing the external environment [7,8].
7
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.
2.2.1 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 mechanisms and pathways (Figure 2.1), 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 simultaneously 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
2.2.1.1. 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
8
monomers into an initially-reversible complex. As protein concentration increases or time
passes, the protein complex may become irreversible and lead to aggregation (Figure
2.1a). Formation of intermolecular disulfide linkages (possibly through disulfide
interchange reactions) is one cause of this irreversibility [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].
9
1
Higher oligomers
(potentially irreversible)
or
Native
protein
Reversible oligomerization of native protein
Higher oligomers
(typically irreversible)
2
Native
protein
Conformational
change / unfolding
Oligomerization of
non-native protein
Higher oligomers
(potentially irreversible)
3
Native
protein
Chemicallymodified protein
Oligomerization of modified protein;
potential recruitment of native proteins
Visible particulates
or precipitation
4
Native
protein
Critical
nucleus
Addition of native proteins onto
nucleation sites, with partial unfolding
Visible aggregates,
adsorption loss
or precipitation
5
Native
protein
Container walls /
air-liquid interface
Adsorption and
surface-induced
partial unfolding
Aggregation of
altered protein
(as in Mechanism 2)
Figure 2.1: Schematic illustration of common mechanisms of aggregation. Multiple
mechanisms may be at work in any given system. Adapted with permission from [5].
Copyright © 2009 Bentham Science Publishers.
Human interleukin-1 receptor anatagonist (IL-1ra) is part of the IL1/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
10
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 also able to
dissociate aggregate particles of insulin, indicating that the association was largely
charge-based [11].
2.2.1.2. 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 (Figure 2.1b). 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
11
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 unimportant. 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.
2.2.1.3. 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 (Figure 2.1c). Chemical changes may profoundly alter protein
12
properties such as solvent accessibility of hydrophobic patches, reduction in electrostatic
repulsion due to modification of charged residues, or disruptions of 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 susceptible 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].
2.2.1.4. Nucleation-dependent aggregation (Mechanism 4)
In contrast to the three previous mechanisms, which are based on
interactions between individual protein molecules, protein aggregation can also be
13
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 (Figure
2.1d). 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 10residue 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 positivelycharged 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
14
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 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 oilwater 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. Polysorbate 20 is known to compete with protein molecules at air-water
and oil-water interfaces (model hydrophobic systems), where it prevents the structural
perturbations and subsequent aggregation of the protein molecules at the unprotected
interfaces [20].
2.2.1.5. 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 (Figure 2.1e). The resulting non-native
conformation then serves as a starting point for aggregation in solution or directly on the
15
surface, as described in Mechanism 2 (above). While the previous mechanisms have dealt
with proteins in solution, this “heterogeneous” 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 interactions 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
concentrations 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 aggregation, 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].
16
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 measured
by activated partial thromboplastin time) of rFVIII. High surfactant concentrations,
however, reduced these surface-induced effects due to competitive hindrance of rFVIII
adsorption at the surfactant-coated surface [24].
2.2.2. Surfactants used in drug products
Surfactants are amphipathic, surface-active molecules that readily adsorb
at interfaces. Although literally thousands of different surfactants are commercially
available, all generally consist of a hydrophilic “head”, which can be ionic or a highly
polar polymer, and a hydrophobic “tail”, often a long-chain aliphatic hydrocarbon group.
Surfactants can be classified as anionic, cationic, nonionic and amphoteric based upon the
nature of the hydrophilic “head”. An excellent example of a widely-used anionic
surfactant is sodium dodecyl sulfate (SDS); the dodecyl (C12) tail is hydrophobic, while
the sulfate group is highly polar. Surfactants have wide-spread applications in industry as
emulsifiers, foaming agents, wetting agents, dispersants and detergents. The
pharmaceutical and biotechnology industries primarily use nonionic surfactants for a
variety of applications (including stabilization of protein therapeutics), because these
surfactants exhibit low toxicity and low sensitivity to the presence of electrolytes.
17
At low concentrations, surfactants will adsorb to all available interfaces,
replacing the higher energy molecules, and lowering the overall interfacial free energy of
the system. However, as more surfactant molecules are introduced, eventually the
interfaces become saturated. At this point, energy reduction is achieved by formation of
micelles or other aggregated states, in which the hydrophobic “tails” are in the center and
away from the surrounding water. The critical micelle concentration (CMC) is defined as
the bulk concentration of surfactant at which micellization begins to occur, and is an
important fundamental property of a surfactant.
However, the CMC does not completely describe the surfactant’s effect
in a protein mixture. If the surfactant has a high affinity for a surface, then surfactant
concentrations near the CMC will tend to stabilize protein against surface-induced
denaturation, even when no specific binding of the surfactant to the protein is observed.
In contrast, if the surfactant stabilizes proteins by directly binding to them, the effective
surfactant concentration is related to the ratio of surfactant to protein, rather than the
CMC [25]. Equilibrium air-water interfacial tensiometry measurements of surfactant
solutions at various concentrations are commonly used to estimate the CMC. In this
approach, the CMC is determined as the bulk surfactant concentration beyond which the
equilibrium interfacial tension is independent of surfactant concentration (i.e. adding
more surfactant has no effect on the interfacial tension). This approach is often applied to
identify the apparent CMC of a surfactant in protein-surfactant mixtures as well. In either
case, it is implicitly assumed that when the CMC of the surfactant is met, the steady-state
interfacial tension is governed entirely by the surfactant at the interface, and independent
of other factors. It is important to note that experimental measures of the CMC are
generally not sharp transitions, and are strongly dependent upon factors such as ionic
18
strength and temperature. Thus, literature values for a given surfactant may vary widely
between reports [26].
2.2.2.1 Polysorbates
O
O
O
w
OH
O
O
x
HO
O
O
z
OH
y
R (Aliphatic)
R=
(CH2)11
(PS-20)
(CH2)7CH=CH(CH2)8 (PS-80)
w + x + y + z = 20
Figure 2.2: 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.
Polysorbates have a common structure consisting of a sorbitan ring with
poly(ethylene oxide) at the hydroxyl positions, and differ only in the structures of the
fatty acid side chains (Figure 2.2). Differences in the length and unsaturation of the fatty
acid side chain structures cause the binding affinities of polysorbates with proteins to
differ [27]. The most commonly-used nonionic surfactants are polysorbate 20
(polyoxyethylene sorbitan monolaureate), sold commercially as Tween 20®, and
polysorbate 80 (polyoxyethylene sorbitan monooleate, or Tween 80®). Polysorbate 80 is
considerably more surface-active and has a lower CMC than polysorbate 20, because it
has a longer and monounsaturated aliphatic chain [28]. Polysorbate 80 also exhibits a
weaker interaction with human serum albumin than polysorbate 20, again due to its
longer fatty acid chain [21, 27]. Differences in the binding affinity and interaction of
polysorbate 20 and 80 with darbepoetin alfa have been reported. Polysorbate 80 binds to
darbepoetin alfa with minimal effect on its tertiary structure, while binding of polysorbate
20 binding caused partial unfolding of the protein [29].
As mentioned above, polysorbates are widely reported to suppress
aggregation upon agitation, shaking, freeze-drying and freeze-thawing processes, and can
19
substantially prevent protein adsorption at solid surfaces [21, 24, 30-33]. However, the
effectiveness appears to depend on the particular stress involved: in one study, stirring of
an antibody suspension was found to be more stressful than shaking, despite the renewal
of air-water interfaces during shaking. Polysorbate 20 at concentrations above 0.0025%
(w/v) inhibited aggregation during shaking. However, at lower polysorbate
concentrations, the protein was destabilized by shaking, and much higher surfactant
concentrations were required to stabilize stirred suspensions [34]. The polysorbates are
susceptible to autoxidation at moderate temperatures, primarily by radical reactions at the
PEO and unsaturation sites of the olefinic moieties, and hydrolysis was observed as a
significant mechanism of degradation at higher temperatures [35]. In another study,
addition of Tween 80® inhibited aggregation of IL-2 mutein during shaking.
Paradoxically, Tween 80® accelerated the aggregation of the same protein in a
temperature-dependent manner during storage. The build-up of peroxides from
autoxidation of degraded polysorbates increased the oxidization rate of the protein,
therefore compromising its stability in storage [36].
2.2.2.2. Poloxamers
Triblock copolymers of the form polyethylene oxide–polypropylene
oxide–polyethylene oxide (PEO–PPO–PEO), or poloxamers (commercially available as
Pluronics® or SynperonicsTM), constitute another class of nonionic surfactant commonly
used in the pharmaceutical industry (Figure 2.3). The poloxamers are listed as
pharmaceutical excipients in the U.S. and British Pharmacopoeia, and have been used
extensively in a variety of pharmaceutical formulations [37]. Poloxamers show complex
aggregation behavior, involving unimers, oligomers, micelles of various geometries, and
larger clusters, with strong temperature and concentration dependences. The critical
micelle temperature and CMC values of such triblocks have been estimated over a wide
20
range of molecular weights and PPO/PEO ratios, by a number of different methods [26,
38-41]. In general, triblocks with a larger hydrophobic (PPO) domain form micelles at
lower concentrations or, at a constant triblock molar concentration, have lower critical
micelle temperatures. For a given PPO:PEO ratio, triblocks of higher molecular weight
form micelles at lower concentrations and temperatures. The size of the hydrophilic PEO
group appears to play a smaller role in the micellization process. Alexandridis et al.
[39Error! Bookmark not defined.] performed a thermodynamic analysis of the formation
of triblock micelles, to obtain standard free energies, enthalpies, and entropies of
micellization for a number of poloxamers. They found that the standard enthalpy of
micellization was positive for all triblocks tested, indicating that the transfer of unimers
from solution to the micelle is an enthalpically unfavorable, endothermic process. A
negative entropy contribution was thus implicated as the driving force for micellization.
Poloxamer 188 (BASF Pluronic® F68, Figure 2.3) is widely used for the
large scale production of mammalian cell culture, and also where bioreactors are
increasingly used to amplify a cell population. It is 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].
However, removing poloxamer 188 during product recovery may compromise the
product yield, as well as inhibit the growth of some cell lines [44].
21
HO
O
H
O
O
79
CH3
26
79
Figure 2.3: 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].
2.2.3. 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 (Figure 2.4). Some surfactants may function
according to only one of these mechanisms, while others may function according to both.
protein
protein
protein
a
b
Figure 2.4: 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.
2.2.3.1. 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
22
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 surfactant-protein
complex in modulating interfacial behavior will provide direction for much-needed
process improvements in the production and finishing of therapeutic proteins.
2.2.3.1.1. 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 complexes and subsequent solubilization of these complexes.
Alternatively, 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
23
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 solidwater 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 sufficiently 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 introduced 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 surfacedependent 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
24
(relative to proteins) is likely to contribute to a reduction in protein adsorption only if the
surfactant-surface affinity is sufficiently high.
2.2.3.1.2. 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 recombinant Factor VIII (rFVIII) was
investigated at a hydrophilic and hydrophobic solid-water interface in the presence of
polysorbate 80 [24]. At the hydrophobic surface, the presence of polysorbate 80 in the
protein solution resulted in reduced protein adsorption, while rFVIII adsorption at the
hydrophilic surface was entirely unaffected by the presence of polysorbate 80. As in the
case of polysorbate 80 and lysozyme, these observations were attributed to the high
binding strength between the surfactant and the hydrophobic surface, and the relatively
low affinity between polysorbate 80 and the hydrophilic surface. 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.
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
25
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 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 surfactantmediated prevention of protein aggregation.
2.2.4. 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.
2.2.4.1. 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 concentration (Figure 2.5)
[57, 58]:
26
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 displacement 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].
27
2
3
4
5
Steady-State
Surface Tension
1
Region 1:
Very low surfactant
concentrations
air
liquid
protein
• Surfactant predominantly
appears as unimers in the
bulk liquid phase
• Limited interactions of
surfactant molecules with
protein and liquid interface
Surfactant Concentration (log10)
Region 2:
Low surfactant
concentrations
air
liquid
protein
• Surfactant unimers appear
at interface and in bulk liquid
Region 5:
Very high surfactant
concentrations (> CMC)
air
• Surfactant molecules begin
to displace protein from the
air-liquid interface
protein
• Appreciable interactions of
surfactant molecules with
liquid interface and protein
liquid
• Substantial interactions of
surfactant molecules with
liquid interface and protein
air
liquid
• Surfactant unimers appear
at interface, in bulk liquid,
and at protein surfaces
• Appreciable interactions of
surfactant molecules with
liquid interface, but limited
interaction with protein
Region 4:
High surfactant
concentrations
Region 3:
Moderate surfactant
concentrations
protein
• Protein completely displaced
from air-liquid interface
• Strong interactions between
surfactant and interface;
protein coated by surfactant
protein
• Micelles form in bulk liquid
Figure 2.5: Schematic illustration of surface tension depression associated with five
regimes of protein-surfactant and surfactant-interface interactions. Redrawn with
permission from [58].
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 exhibited 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
28
adsorption is evident, or if the steady-state interfacial behavior is 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 (Figure 2.6). 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 Tension (γ)
surface was dominated by the surfactant [22].
Low [S]
S only
S+P
Intermediate [S]
High [S]
S only
S+P
S/S+P
Time
Figure 2.6: 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].
2.3. A testable, thermodynamic argument to guide surfactant selection
2.3.1. 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
29
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 (Figure 2.7).
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 proteinfree 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 (Figure 2.7).
Air
Water
Figure 2.7: 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.
30
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:
rate
= km ⋅ c n ,
(1)
where n, the order of the reaction, was obtained from measurements of the initial
adsorption rate at each concentration, c, in the bulk phase.
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:
E 1
ln km =
−  a ⋅  + ln A ,
 R T
(2)
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 airwater interface) and an activated complex. From this model, the rate constant can be
described in thermodynamic terms:
k T
km =  b
 h
 −∆G RT
,
e

(3)
where kb and h are Boltzmann’s and Planck’s constants, respectively, and ΔG is the
Gibbs free energy of transition. Thus, since ∆G = ∆Η − Τ∆S ,
k 
 k ∆S 
 ∆H 1 
−
⋅  + ln  b +
ln  m  =

R 
 R T
T 
h
(4)
31
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 (Figure 2.7), 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 intermediate. 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 (Figure 2.6). This kind of “synergistic” effect is well documented for synthetic
polymer-surfactant mixtures [61], but less well understood in relation to proteinsurfactant 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). 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 aggregation and adsorption loss.
32
2.3.2. 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 surfactant
aggregate are based on entropy, resulting from the highly-ordered clathrate cages of
water molecules that surround a nonpolar molecule in an aqueous environment. A
thermodynamic 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 speculate 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 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 (Figure 2.8). 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.
33
air
water
+
air
water
+
+
protein
protein
protein
Figure 2.8: 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).
These arguments may be more significant in relation to associations of
large-molecule surfactants (e.g. poloxamers), as opposed to the smaller 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, yet a
synergistic effect in the kinetics of surface tension depression similar to that described in
Section 2.4.1 has been recorded for mixtures of selected proteins and poloxamers 188
(Pluronic® F68). As with the polysorbates, surface tension depression in a poloxamerprotein mixture tends to be faster than for the same concentration of the surfactant alone
(Figure 2.6).
34
2.3.3 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 hydrophilic
poloxamer (80% PEO) in various sodium salt solutions. While the triblock did not form
micelles in pure water at ambient temperatures, it exhibited aggregation and micellization
in salt solutions. The cloud point and critical micelle temperature (CMT) decreased
substantially in the presence of salts. They found that the average micelle size increased
with salt concentration, and the relative effect of the different anions to enhance
micellization followed the Hofmeister series (i.e. PO43– > SO42– > F– > Cl– > Br–),
consistent 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 commonly associated with triblocks
and other nonionic surfactants in water. It is due in part to changes in hydrogen bonding
and increasingly attractive interactions between the PEO chains as the temperature
increases, making the water a “less good” solvent [67]. The influence of salts can be
35
explained in a similar fashion, being primarily caused by the existence of a salt-deficient
zone surrounding the PEO chains: small ions with little polarizability would be repelled
by the poorly polarizable PEO chain. This salt-deficient zone gives rise to an attractive
component in the interaction between PEO segments. As polymer segments approach
each other, their salt depletion zones overlap and the surrounding water molecules are
liberated to the bulk solution. The excluded water molecules have a lower chemical
potential 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-enriched 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 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 of higher ionic
strength, and be driven to associate with the surface by a “salting out” mechanism. Thus,
surfactants could effectively stabilize a protein by association with the surface, forming a
surfactant-protein complex that guards against close approach and subsequent
aggregation with other proteins. Conversely, the surface at an air-water interface is
depleted of salts relative to the bulk solution [70], and surfactant unimers at the surface
would encounter a “more watery” micro-environment, and would thus not adsorb with
high affinity to the interface. The effects of these microscopic changes in ionic strength
are consistent with the accelerated surface tension depression observed for surfactantprotein mixtures (relative to protein-free surfactant), and also with protein stabilization in
the vicinity of the air-water interface, even when the interface is only partially occupied
by surfactant. Further research into these mechanisms is clearly warranted.
36
2.4. Conclusions
Surfactants stabilize proteins by their preferential location at an interface
and/or their association with protein in solution. In the common case of surfactant-protein
interaction governed by hydrophobic association, the molecular origins of surfactantmediated stabilization of protein can be inferred, in part, from quantitative models.
Insights can be gained by comparison of thermodynamic barriers that limit surfactant
adsorption, from surfactant-protein mixtures and protein-free surfactant solutions, to the
air–water interface. These barriers are defined by the enthalpy and entropy of transition
from free (whether unimeric or aggregated) surfactant molecules to adsorbed surfactants,
via formation of a rate-limiting structural intermediate. This is easily done, for example,
through analysis of surface tension kinetic data recorded at different temperatures.
Concentration effects on the aggregation state of a surfactant, effects of salt on
micellization, and the thermodynamic barriers to its adsorption are particularly important
factors in determining the effectiveness of a given surfactant at stabilizing a protein. A
fundamental understanding of the mechanisms of aggregation and how surfactants
interact with interfaces and proteins (particularly the preferential location of a surfactant
at an interface, or its association with protein in solution), provides guidance in selecting
surfactants and excipients to reduce protein losses in a given application.
37
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42
PROTEIN EFFECTS ON SURFACTANT ADSORPTION SUGGEST THE
DOMINANT MODE OF SURFACTANT-MEDIATED STABILIZATION OF
PROTEIN
Hyo Jin Lee,a,b Arnold McAuley,b and Joseph McGuirea,*
a
School of Chemical, Biological and Environmental Engineering, Oregon State University,
Corvallis, OR 97331
b
Proces and Product Development Department Amgen Inc., Thousand Oaks, CA 91320
*
Corresponding Author:
Joseph McGuire
School of Chemical, Biological and Environmental Engineering
Oregon State University
103 Gleeson Hall
Corvallis, OR 97331-2702
Tel:
541-737-6306
Fax:
541-737-4600
Email: mcguirej@engr.orst.edu
43
CHAPTER 3
PROTEIN EFFECTS ON SURFACTANT ADSORPTION SUGGEST THE
DOMINANT MODE OF SURFACTANT-MEDIATED STABILIZATION OF
PROTEIN
Abstract
Nonionic surfactants are commonly used to stabilize proteins in upstream
and downstream processes and drug formulation. However, the effectiveness of a
surfactant strongly depends on its mechanism(s) of action but selection of surfactants for
protein stabilization currently is not made with benefit of any quantitative, predictive
information to ensure that this requirement is met. Often the kinetics of surface tension
depression (by surfactant) in protein-surfactant mixtures tends to be greater than that
recorded for surfactant alone at the same concentration. Here, we compare surface
tension depression by poloxamer 188 (Pluronic® F68), polysorbate 80 (PS80), and
polysorbate 20 (PS20) in the presence and absence of lysozyme and recombinant protein,
at different surfactant concentrations and temperatures. The kinetic results are interpreted
with reference to a mechanism for surfactant adsorption governed by the formation of a
rate-limiting structural intermediate (i.e., an “activated complex”) between surfactant
aggregates and protein. The presence of lysozyme was seen to increase the rate of
surfactant adsorption in relation to surfactant acting alone at the same concentrations for
the polysorbates while less of an effect was seen for Pluronic® F68. However, the
addition of salt was observed to accelerate the surface tension depression of Pluronic®
F68 in the presence of lysozyme. Findings of aggregate present in polysorbates even
below the CMC and Pluronic® F68 displaying greater aggregation and micellization in
salt solutions have lead us to believe that an interaction between lysozyme and surfactant
44
aggregates may cause aggregate destabilization and an increased availability of surfactant
monomers for adsorption. The addition of a more hydrophobic, surface active protein in
place of lysozyme resulted in greater enhancement of surfactant adsorption than that
recorded in the presence of lysozyme. A simple thermodynamic analysis indicated the
presence of protein caused a reduction in ∆𝑮 for the surfactant adsorption process, with
this reduction deriving entirely from a reduction in ∆𝑯. We suggest that protein
accelerates the adsorption of these surfactants by disrupting their self associations,
releasing surfactant monomers.
3.1 Introduction
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 [1]. Several workers have reported on the different
mechanisms of aggregation, and suggested possible methods to inhibit aggregation [2-4].
Several mitigation strategies are used in the biotechnology industries to stabilize
therapeutic proteins, but the addition of surfactants appears to be a general approach [5].
Surfactants are able to stabilize proteins through two major mechanisms.
One involves the preferential location of surfactant at nearby interfaces, in this way
precluding protein adsorption [6]. The other mechanism involves association of protein
and surfactant molecules into complexes that prevent proteins from interacting with
surfaces as well as each other [7]. In general, both mechanisms must be at play for
effective protein stabilization against aggregation and activity loss, but selection of
45
surfactants for protein stabilization currently is not made with benefit of any quantitative,
predictive information to ensure that this requirement is met.
An interesting observation characteristic of surface tension depression by
surfactant-protein mixtures is that the kinetics of surface tension depression (by
surfactant) in such mixtures often tends to be greater than that recorded for surfactant
alone at the same concentration [8]. It is reasonable to expect that such “accelerated
adsorption” of surfactant in the presence of protein is governed by surfactant-protein
interactions and must therefore be a function of parameters important to protein
stabilization by that surfactant. We recently hypothesized that parameters determining
whether surfactant stabilization occurs mainly by association with the interface or by
association with protein ought to be quantifiable for different surfactants by analysis of
such accelerated adsorption data [9]. In particular, the adsorption of surfactant monomers
may be enhanced by interaction between surfactant aggregates and protein near the
interface, leading to the release of monomers for adsorption. As depicted in Figure 3.1
(top), transport of monomers from an aggregated state to the air-water interface in the
absence of protein would require that they dissociate from the aggregate and migrate
through the liquid in order to adsorb. The presence of protein may reduce the major
enthalpic barrier associated with surfactant dissociation, and/or reduce the major entropic
barrier faced by the surfactant in moving from the aggregate to the interface [10]. The
protein may facilitate aggregate disruption, leading either to an increased concentration of
surfactant monomers thus enhancing adsorption (Figure 3.1, middle), or to the formation
of stable, surfactant-protein complexes having little or no effect on surfactant adsorption
rate (Figure 3.1, bottom). Thus we expect that accelerated surfactant adsorption in the
presence of protein will occur with surfactants that stabilize protein mainly by their own
46
adsorption at interfaces, and we expect the absence of accelerated surfactant adsorption
will occur with surfactants that form stable surfactant-protein associations.
air
water
+
air
water
+
protein
protein
protein
air
water
protein
protein
protein
Figure 3.1: In the absence of protein (top), surfactant monomers must dissociate and
migrate through the liquid in order to adsorb. The presence of protein may facilitate
aggregate disruption, leading either to an increased concentration of surfactant monomers
thus enhancing adsorption (middle), or to the formation of stable, surfactant-protein
complexes having little or no effect on surfactant adsorption rate (bottom).
In this paper we complete the first step in testing the hypothesis outlined
above, through comparison of surface tension depression by selected surfactants in the
presence and absence of protein, at different surfactant concentrations and temperatures.
The kinetic results are interpreted with reference to a mechanism for surfactant
adsorption governed by the formation of a rate-limiting structural intermediate (i.e., an
“activated complex”) between surfactant aggregates and protein.
47
3.2 Materials and Methods
3.2.1. Proteins and surfactants
The proteins used were lyophilized chicken egg white lysozyme (Sigma
Aldrich, Saint Louis, MO) and an Amgen recombinant protein. Lysozyme was dissolved
in Dulbecco’s phosphate buffered saline (1X PBS, Invitrogen, Carlsbad, CA) at 100
mg/mL or higher and was filtered using a 0.2 um PVDF filter (Millipore, Billerica, MA)
before the surface tension experiments. The final concentration of lysozyme used in the
measurements was 5 mg/mL. Amgen recombinant protein is 18.8 kDa that consists of
174 amino acids with 2 disulfide bonds. Recombinant protein was provided in 10 mM
sodium acetate, 5% sorbitol at pH 3.5 at 3 mg/mL and was concentrated to 20 mg/mL
using Amicon stir cells (Millipore, Billerica, MA) and filtered using a 0.2um PVDF filter
before use. The final concentration of the recombinant protein used in the measurements
was 1mg/mL.
The surfactants studied were Poloxamer 188 (Pluronic ®F68, BASF,
obtained from Sigma Aldrich, Saint Louis, MO) polysorbate 80 (PS 80, Croda, East
Yorkshire, England) and polysorbate 20 (PS 20, Croda, East Yorkshire, England). Stock
solutions at high concentrations of the surfactants were made in 1X PBS and in 10 mM
sodium acetate, 5% sorbitol at pH 3.5 buffers and diluted to the respective buffers to have
a range of concentrations. Buffer salts and sorbitol were from obtained from JT Baker
(Philipsburg, NJ)
3.2.2. Surface tension measurements
Surface tension was measured using a 1-cm wide Wilhelmy plate (Kruss,
Hamburg, Germany) on a Kruss K100 tensiometer (Hamburg , Germany). 19 mL of
buffer was placed in a 50 mL glass vessel housed within a temperature controlled
jacketed stage and surface tension was recorded every 10 seconds for 220 seconds. Then,
48
1 mL of the desired sample (which could be the protein or surfactant by itself or
surfactant-protein mixture) was introduced and stirred for 4 seconds before measurement
started again and recorded every 10 seconds for a total of 220 seconds and repeated 3
times. In order to capture the initial surface tension depression kinetics, only the last data
point of the buffer is shown at time zero. Most of the experiments were done at room
temperature. However to study the effect of temperature on surface tension depression,
additional experiments were performed at 5 °C and 40 °C.
3.3 Results and Discussion
3.3.1. Effect of surfactant concentration
Representative plots of the early kinetics of surface tension depression
are shown in Figures 3.2-3.4 for selected concentrations of PS 80 (Figure 3.2), PS 20
(Figure 3.3), and Pluronic ® F68 (Figure 3.4), in the presence and absence of lysozyme.
Both the initial rate and extent of surface tension depression generally increased with
increasing surfactant concentration. Lysozyme alone at 5 mg/mL displayed less surface
tension depression (about 64 mN/m after 220 s) than any of the surfactants at surfactant
concentrations of 5 ppm and above. Thus the plots of Figures 3.2-3.4 suggest that for
lysozyme-surfactant mixtures, interfacial behavior was governed predominantly by
surfactant at concentrations greater than 5 ppm.
49
75
Surface Tension (mN/m)
70
5 ppm PS 80
5 ppm PS 80 +Lysozyme
10ppm PS 80
10 ppm PS 80 +Lysozyme
20 ppm PS 80
20 ppm PS 80 +Lysozyme
50 ppm PS 80
50 ppm PS 80 +Lysozyme
100 ppm PS 80
100 ppm PS 80 +Lysozyme
5 mg/mL Lysozyme
65
60
55
50
45
40
0
50
100
150
200
250
Time (sec)
Figure 3.2: Adsorption isotherm (surface tension vs. time) of PS 80 and lysozyme
mixture at 25 °C in 1X PBS.
75
Surface Tension (mN/m)
70
5 ppm PS 20
5 ppm PS 20 +Lysozyme
10 ppm PS 20
10 ppm PS 20 +Lysozyme
20 ppm PS 20
20 ppm PS 20 +Lysozyme
50 ppm PS 20
50 ppm PS 20 +Lysozyme
100 ppm PS 20
100 ppm PS 20 +Lysozyme
5 mg/mL Lysozyme
65
60
55
50
45
40
35
0
50
100
150
200
250
Time (sec)
Figure 3.3: Adsorption isotherm (surface tension vs. time) of PS 20 and lysozyme
mixture at 25 °C in 1X PBS.
50
75
Surface Tension (mN/m)
70
5 ppm F68
5 ppm F68 +Lysozyme
10 ppm F68
10 ppm F68 +Lysozyme
20 ppm F68
20 ppm F68 +Lysozyme
50 ppm F68
50 ppm F68 +Lysozyme
500 ppm F68
500 ppm F68 +Lysozyme
5 mg/mL Lysozyme
65
60
55
50
45
0
50
100
150
200
250
Time (sec)
Figure 3.4: Adsorption isotherm (surface tension vs. time) of Pluronic® F68 and
lysozyme mixture at 25 °C in 1X PBS.
For PS 80 and PS 20 at the lowest concentrations shown (5 and 10 ppm),
the presence of lysozyme was seen to increase the rate of surfactant adsorption in relation
to surfactant acting alone at the same concentrations. Using the dye 1,6-diphenyl 1,3,5hexatriene, Kerwin [13] recorded initiation of aggregate formation in solutions of
polysorbates 20 and 80 at concentrations well below the CMC of each surfactant, with PS
80 showing more pronounced aggregation than PS 20. In such tests, an increase in
fluorescence is detected when the (hydrophobic) dye partitions from the aqueous phase
into the apolar phase of an aggregate interior [11,12]. Both polysorbates showed initiation
of aggregate formation in that work at concentrations consistent with those used in
construction of Figures 3.2-3.3. The enhanced surfactant adsorption recorded in the
presence of lysozyme (Figures 3.2 and 3.3) is thus qualitatively consistent with an
interaction between lysozyme and surfactant aggregates causing aggregate destabilization
and an increased availability of surfactant monomers for adsorption. It is also consistent
with the observation that accelerated surfactant adsorption is more evident for PS 80 than
51
for PS 20 in these cases. At polysorbate concentrations of 20 ppm and above, no
appreciable difference was recorded between lysozyme-surfactant mixtures and the
surfactant alone at the same surfactant concentration. This suggests that the population of
surfactant monomers near the interface is sufficiently high (and the strength of proteinaggregate association sufficiently weak), such that adsorption is no longer rate-limited by
the protein-aggregate structural intermediate.
On the other hand, lysozyme showed much less of an effect on
Pluronic® F68 adsorption. This would be consistent with Pluronic® F68 undergoing no
appreciable aggregate formation under these conditions. Pluronic® F68 is not expected to
form micelles in water except at very high concentrations [14-17]. Like other Pluronics®
it shows decreased solubility in the presence of salts. Thus we would expect lysozyme to
show a more pronounced effect on Pluronic® F68 adsorption in the presence of salt.
3.3.2. Effect of salt
While Pluronic® F68 would exhibit greater aggregation and
micellization in salt solutions, the surface at an air-water interface is depleted of salts
relative to the bulk solution [18], and surfactant monomers would thus not be expected to
adsorb with high affinity to the interface. Figure 3.5 shows a negligible effect on
adsorption of Pluronic® F68 in the absence of lysozyme, upon addition of 250 mM NaCl.
However, the addition of salt did accelerate the surface tension depression of Pluronic®
F68 in the presence of lysozyme, albeit modestly, consistent with an increased population
of Pluronic® F68 aggregates in solution. Even at 50 ppm, where Figures 3.2-3.4 indicate
the adsorption is apparently not rate-limited by the protein-aggregate structural
intermediate for any of the three surfactants studied, the addition of salt was observed to
accelerate the surface tension depression of Pluronic® F68 in the presence of lysozyme
52
(Figure 3.6). No effect upon addition of salt was evident for adsorption of PS 80 or PS 20.
This is consistent with prior reports of polysorbate aggregation behavior being largely
unaffected by the presence of salts [13,19].
76
74
Surface tension (mN/m)
72
70
68
10 ppm F68 0 mM NaCl
10 ppm F68 +Lysozyme 0 mM NaCl
10 ppm F68 250 mM NaCl
10 ppm F68 +Lysozyme 250 mM NaCl
66
64
62
60
58
56
54
0
50
100
150
200
250
Time (sec)
Figure 3.5: Effect of salt on the adsorption isotherm (surface tension vs. time) of 10 ppm
Pluronic® F68 and lysozyme mixture
75
Surface tension (mN/m)
70
65
50 ppm F68 250 mM NaCl
50 ppm F68 +Lysozyme 250 mM NaCl
50 ppm PS 80 250 mM NaCl
50 ppm PS 80 + Lysozyme 250 mM NaCl
50 ppm PS 20 250 mM NaCl
50 ppm PS 20 + Lysozyme 250 mM NaCl
60
55
50
45
40
0
50
100
150
200
250
Time (sec)
Figure 3.6: Effect of salt on the adsorption isotherm (surface tension vs. time) of
surfactants (50 ppm Pluronic® F68, PS 80 and PS 20) and lysozyme mixture
3.3.3. Effect of temperature and comparison to a simple model for protein-mediated
acceleration of surfactant adsorption
53
The early kinetics of surface tension depression were recorded for each
surfactant at 5 and 10 ppm, in the presence and absence of lysozyme, at 5, 25, and 40 °C.
Representative plots (constructed for surfactant concentrations of 5 ppm) are shown in
Figures 3.7-3.9 for PS 80 (Figure 3.7), PS 20 (Figure 3.8), and Pluronic® F68 (Figure
3.9). Hydrophobic interaction is enhanced with increasing temperature, and for each
surfactant, accelerated surfactant adsorption in the presence of protein is seen to increase
with temperature. Again, this effect is seen to be clearly more pronounced for the
polysorbates than for Pluronic® F68.
80
Surface tension (mN/m)
75
70
5 ppm PS 80 5C
5 ppm PS 80 +Lysozyme 5C
5 ppm PS 80 25C
5 ppm PS 80 +Lysozyme 25C
5 ppm PS 80 40C
5 ppm PS 80 +Lysozyme 40C
65
60
55
50
0
50
100
150
200
250
Time (sec)
Figure 3.7: Adsorption isotherm (surface tension vs. time) of 5 ppm PS 80 and lysozyme
mixture at 5 °C, 25 °C, and 40 °C in 1X PBS.
54
80
Surface tension (mN/m)
75
70
5ppm PS 20 5C
5ppm PS 20 +Lysozyme 5C
5ppm PS 20 25C
5ppm PS 20 +Lysozyme 25C
5ppm PS 20 40C
5ppm PS 20 +Lysozyme 40C
65
60
55
50
0
50
100
150
200
250
Time (sec)
Figure 3.8: Adsorption isotherm (surface tension vs. time) of 5 ppm PS 20 and lysozyme
mixture at 5 °C, 25 °C, and 40 °C in 1X PBS.
80
Surface tension (mN/m)
75
70
5 ppm F68 5C
5 ppm F68 +Lysozyme 5C
5 ppm F68 25C
5 ppm F68 +Lysozyme 25C
5 ppm F68 40C
5 ppm F68 +Lysozyme 40C
65
60
55
50
0
50
100
150
200
250
Time (sec)
Figure 3.9: Adsorption isotherm (surface tension vs. time) of 5 ppm Pluronic ® F68 and
lysozyme mixture at 5 °C, 25 °C, and 40 °C in 1X PBS.
It is instructive to invoke classical transition-state theory [20] in order to
consider the effect of temperature in terms of an equilibrium between the “reactants” in
this case (i.e., surfactant aggregates and dissolved protein) and the protein-surfactant
activated complex (i.e., the structural intermediate described with reference to Figure 3.1).
55
For this purpose, the initial decrease in surface tension (𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑟𝑎𝑡𝑒) at each temperature
and surfactant concentration (𝐶) tested can be represented as
𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑟𝑎𝑡𝑒 = 𝑘𝑚 ∙ 𝐶 𝑛 ,
where 𝑘𝑚 is the rate constant characterizing the initial decrease in surface tension and 𝑛
is the order of the reaction. In order to determine the initial rate in each case, surface
tension vs. time (𝑡) data were fit to a simple three-parameter exponential decay function
𝑓(𝑡) = 𝑎𝑒
�
𝑏
�
𝑡+𝑐
and initial slopes calculated. The rate constant and reaction order are then easily
determined by analysis of plots of 𝑙𝑛 (𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑟𝑎𝑡𝑒) vs. 𝑙𝑛 𝐶 for each surfactant at each
temperature. The activation energy, 𝐸𝑎 , for adsorption in each case can then be derived
from the slope of a plot of ln 𝑘𝑚 vs. 1/𝑇, according to the Arrhenius equation:
𝑙𝑛 𝑘𝑚 = − �
𝐸𝑎 1
∙ � + 𝑙𝑛 𝐴,
𝑅 𝑇
where 𝑅 is the gas constant, 𝑇 is temperature, and 𝐴 is the Arrhenius pre-exponential
factor (Figure 3.10).
56
20
18
ln km
F68
F68 + Lysozyme
PS 80
PS 80 + Lysozyme
PS 20
PS 20 + Lysozyme
10
8
6
4
0.0031
0.0032
0.0033
0.0034
1/T, °C
0.0035
0.0036
0.0037
-1
Figure 3.10: Arrhenius plot of adsorption during initial surface tension depression. The
energy of activation, 𝐸𝑎 , was derived from the slope of linear regressions.
Considering the effect of temperature in terms of an equilibrium between
reactants and an activated complex, the rate constant can be described in thermodynamic
terms:
𝑘𝑚 =
𝑘𝑏 𝑇
ℎ
∙𝑒
−∆𝐺�
𝑅𝑇 ,
where kb and h are Boltzmann’s and Planck’s constants, respectively, and ΔG is the free
energy of transition. Thus, since ∆𝐺 = ∆𝐻 − 𝑇 ∙ ∆𝑆,
𝑙𝑛
𝑘𝑚
𝑇
=−
∆𝐻
𝑅
1
∙ + �𝑙𝑛
𝑇
𝑘𝑏
ℎ
+
∆𝑆
𝑅
�.
The slope and intercept of plots of 𝑙𝑛 𝑘𝑚 ⁄𝑇 vs. 1⁄𝑇 therefore provide
estimates of the enthalpy (ΔH) and entropy (ΔS) of the transition (Figure 3.11). ∆𝐺 can
thus be calculated at any desired temperature. Table 3.1 lists the thermodynamic
parameters calculated in this fashion for surfactant solutions in the presence and absence
of dissolved protein. A reduction in ∆𝐺 was observed in each case upon addition of
57
protein, with the reduction being more pronounced for the polysorbates than for
Pluronic® F68. With increases observed in the entropic contribution to free energy
(−𝑇 ∙ ∆𝑆) in the presence of protein for each surfactant tested, the entries in Table 3.1
indicate the reduction in ∆𝐺 observed in the presence of protein resulted entirely from a
reduction in ΔH. The likely source for this reduction would be an alteration of the van der
Waals interactions among the regions on the surfactant molecules mediating their selfassociation. Separation of surfactant monomers from aggregates would disrupt van der
Waals interactions and produce an unfavorable enthalpy. Thus based on this analysis we
suggest that protein is accelerating the adsorption of the surfactants not by reducing the
entropic barrier faced by the surfactant in moving from the aggregate to interface, but by
disrupting the surfactant self-association.
16
14
F68
F68
F68++Lysozyme
Lysozyme
F68
PS
PS80
80
PS
PS80
80++Lysozyme
Lysozyme
PS
PS20
20
PS
PS20
20++Lysozyme
Lysozyme
ln (km/T)
12
6
4
2
0
0.0031
0.0032
0.0033
0.0034
1/T, °C
0.0035
0.0036
0.0037
-1
Figure 3.11: Determination of thermodynamic components of the activation barrier. The
slope and intercept of linear regression provide basis for calculating ∆𝐻 and ∆𝑆,
respectively.
58
reaction order
Ea(kJ/(mol*K))
delG at 40C (kJ/mole)
Enthalpy(H) (kJ/mole)
Entropy(S) (J/(K*mole)
-TdelS at 40C
F68
2.59
17.75
27.97
15.31
-40.47
12.67
F68+Lysozyme
2.65
10.00
26.98
7.56
-62.07
19.43
PS 80
1.64
56.40
54.13
53.96
-0.56
0.18
PS80 + Lysozyme
1.77
34.12
51.50
31.68
-63.35
19.83
PS20
1.75
25.79
52.91
23.35
-94.47
29.57
PS20+Lysozyme
1.81
15.20
51.46
12.75
-123.66
38.71
Table 3.1: Thermodynamic parameters for adsorption of Pluronic® F68, PS 80 and PS
20 in the absence and presence of lysozyme
3.3.4. Effect of protein
Lysozyme is structurally stable and highly cationic under the solution
conditions used here. Based on the analysis above, we would expect that addition of a
more hydrophobic, surface active protein in place of lysozyme would result in greater
enhancement of surfactant adsorption than that recorded in the presence of lysozyme.
Figures 3.12-3.14 show the initial kinetics of surface tension depression recorded for
selected concentrations of PS 80 (Figure 3.12), PS 20 (Figure 3.13), and Pluronic® F68
(Figure 3.14), in the presence and absence of the recombinant protein. At 1 mg/mL, the
recombinant protein reduced surface tension to about 54 mN/m after 220 s, while
lysozyme had reduced surface tension to only 64 mN/m (at 5 mg/mL) after the same time
period. The recombinant protein alone thus displayed more surface tension depression
than any of the surfactants at concentrations of 20 ppm and below (Figures 3.2-3.4).
Considering only surfactant concentrations where surface tension depression by the
surfactant alone is greater than that due to the recombinant protein alone, Figures 3.12
and 3.13 show the adsorption rate was enhanced for each polysorbate by the presence of
the recombinant protein at higher concentrations (50 and 100 ppm) than observed for
lysozyme. The effect of added protein was less apparent in the case of Pluronic® F68. As
described above with reference to lysozyme, these results are consistent with recombinant
protein facilitating aggregate disruption, leading either to an increased concentration of
59
surfactant monomers enhancing adsorption (in the case of PS 20 and PS 80), or to the
formation of stable, surfactant-protein complexes having little or no effect on surfactant
adsorption rate (in the case of Pluronic® F68). Also shown in Figures 3.12-3.14 is the
initial kinetics of surface tension depression recorded for each surfactant at 10 ppm, in
the presence and absence of the recombinant protein. At 10 ppm, surfactant adsorption is
substantially slower than recombinant protein adsorption, but at this surfactant
concentration, surfactant-protein mixtures still show greater surface tension depression
than either component acting alone (in the case of the polysorbates, Figures 3.12 and
3.13). While we must expect that the surface is dominated by protein in these cases, we
hold the enhanced rate recorded is the result of an increased availability of surfactant
monomers at the interface owing to protein-mediated disruption of polysorbate
aggregates.
75
Surface Tension (mN/m)
70
65
10 ppm PS 80
10 ppm PS 80 + rec.protein
50 ppm PS 80
50 ppm PS 80 + rec.protein
100 ppm PS 80
100 ppm PS 80 + rec.protein
rec.protein
60
55
50
45
40
0
50
100
150
200
250
Time (sec)
Figure 3.12: Adsorption isotherm (surface tension vs. time) of PS80 and recombinant
protein mixture at 25 °C in 10 mM sodium acetate, 5% sorbitol, pH 3.5
60
75
Surface tension (mN/m)
70
65
10 ppm PS 20
10 ppm PS 20 + rec.protein
50 ppm PS 20
50 ppm PS 20 + rec.protein
100 ppm PS 20
100 ppm PS 20 + rec.protein
rec.protein
60
55
50
45
40
0
50
100
150
200
250
Time (sec)
Figure 3.13: Adsorption isotherm (surface tension vs. time) of PS 20 and recombinant
protein mixture at 25 °C in 10 mM sodium acetate, 5% sorbitol, pH 3.5
75
Surface tension (mN/m)
70
65
10 ppm F68
10 ppm F68 + rec.protein
50 ppm F68
50 ppm F68 + rec.protein
100 ppm F68
100 ppm F68 + rec.protein
rec.protein
60
55
50
45
40
0
50
100
150
200
250
Time (sec)
Figure 3.14. Adsorption isotherm (surface tension vs. time) of Pluronic® F68 and
recombinant protein mixture at 25 °C in 10 mM sodium acetate, 5% sorbitol, pH 3.5
3.4 Conclusions
We have considered protein effects on surface tension depression by
surfactants commonly used in biopharma, in relation to a mechanism for surfactant
61
adsorption governed by the formation of a rate-limiting structural intermediate (i.e., an
“activated complex” comprised of surfactant aggregates and protein). A simple
thermodynamic analysis indicated the presence of protein caused a reduction in ∆𝑮 for
the surfactant adsorption process, with this reduction deriving entirely from a reduction in
∆𝑯. We suggest that protein accelerates the adsorption of these surfactants by disrupting
their self associations, releasing surfactant monomers. The increased concentration of
surfactant monomers may promote surfactant adsorption, or the formation of stable,
surfactant-protein complexes having little or no effect on surfactant adsorption rate.
Based on this, we expect that accelerated surfactant adsorption in the presence of protein
will occur with surfactants that stabilize protein mainly by their own adsorption at
interfaces, and we expect the absence of accelerated surfactant adsorption will occur with
surfactants that form stable surfactant-protein associations. This expectation is currently
being tested with the proteins and surfactants used here, by revealing the dominant mode
of surfactant-mediated stabilization of protein at solid surfaces, and will constitute the
subject of a future report.
62
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63
[17] S. Borbély, and J.S. Pedersen, Temperature-induced aggregation in aqueous
solutions of pluronic PLURONIC ® F68 triblock copolymer containing small amount of
o-xylene. Physica B: Condensed Matter 276–278 (2000) 363-364.
[18] Y. Levin, A.P. dos Santos, and A. Diehl, Ions at the air-water interface: an
end to a hundred-year-old mystery? Phys Rev Lett 103 (2009) 257802.
[19] K.R. Acharya, S.C. Bhattacharyya, and S.P. Moulik, Effects of
carbohydrates on the solution properties of surfactants and dye–micelle complexation.
Journal of Photochemistry and Photobiology A: Chemistry 122 (1999) 47-52.
[20] O. Levenspiel, Chemical reaction engineering, Wiley, New York, 1999.
64
DOMINANT MODE OF SURFACTANT-MEDIATED STABILIZATION OF
PROTEIN AT SOLID SURFACES
Hyo Jin Lee,a,b Arnold McAuley,b and Joseph McGuirea,*
a
School of Chemical, Biological and Environmental Engineering, Oregon State University,
Corvallis, OR 97331
b
Proces and Product Development Department Amgen Inc., Thousand Oaks, CA 91320
*
Corresponding Author:
Joseph McGuire
School of Chemical, Biological and Environmental Engineering
Oregon State University
103 Gleeson Hall
Corvallis, OR 97331-2702
Tel:
541-737-6306
Fax:
541-737-4600
Email: mcguirej@engr.orst.edu
65
CHAPTER 4
DOMINANT MODE OF SURFACTANT-MEDIATED STABILIZATION OF
PROTEIN AT SOLID SURFACES
Abstract
In earlier work we considered protein effects on surface tension
depression by the surfactants polysorbate 20, polysorbate 80, and Pluronic® F68, in
relation to a mechanism for surfactant adsorption governed by the formation of a ratelimiting structural intermediate Based on results of that work, we proposed that
accelerated surfactant adsorption in the presence of protein (observed with PS 20 and PS
80) will occur with surfactants that stabilize protein mainly by their own adsorption at
interfaces, and that the absence of accelerated surfactant adsorption (observed with F68)
will occur with surfactants that form stable surfactant-protein associations. Optical
waveguide lightmode spectroscopy was used to test this expectation. Adsorption kinetics
were recorded for surfactants (PS 20, PS 80, or F68) and protein (lysozyme or Amgen
recombinant protein) at a hydrophilic solid (SiO2-TiO2) surface. Experiments were
performed in sequential and competitive adsorption modes, enabling the adsorption
kinetic patterns to be interpreted in a fashion revealing the dominant mode of surfactantmediated stabilization of protein in each case. Kinetic results confirmed predictions based
on our earlier quantitative analysis of protein effects on surface tension depression by
surfactants. In particular, PS 20 and PS 80 are able to inhibit protein adsorption only by
their preferential location at the interface, and not by formation of less surface active,
protein-surfactant complexes. On the other hand, F68 clearly is able to inhibit protein
adsorption by formation of protein-surfactant complexes, and not by its preferential
location at the interface.
66
4.1 Introduction
In the previous chapter we considered protein effects on surface tension
depression by the surfactants polysorbate 20, polysorbate 80, and Pluronic® F68, in
relation to a mechanism for surfactant adsorption governed by the formation of a ratelimiting structural intermediate (i.e., an “activated complex” comprised of surfactant
aggregates and protein). A simple thermodynamic analysis indicated the presence of
protein caused a reduction in ∆𝑮 for the surfactant adsorption process (and consequent
acceleration of surface tension depression), with this reduction deriving entirely from a
reduction in ∆𝑯. We suggested that in cases where protein accelerated the adsorption of
the surfactants (observed with PS 20 and PS 80); it did so by disrupting their self
associations, releasing surfactant monomers. The increased concentration of surfactant
monomers then promoted surfactant adsorption. The absence of accelerated surface
tension depression (observed with F68) was attributed to the newly released monomers
participating in the formation of stable, surfactant-protein complexes thus having little or
no effect on surfactant adsorption rate.
Based on that work, we would expect that accelerated surfactant
adsorption in the presence of protein will occur with surfactants that stabilize protein
mainly by their own adsorption at interfaces, and would expect the absence of accelerated
surfactant adsorption will occur with surfactants that form stable surfactant-protein
associations. In this paper we use optical waveguide lightmode spectroscopy to test this
expectation. Adsorption kinetics were recorded for surfactants (PS 20, PS 80, or F68) and
protein (lysozyme or Amgen recombinant protein) at a hydrophilic solid (SiO2-TiO2)
surface. The hydrophilic surface was chosen to enable study of adsorption kinetics in
cases for which protein-surface affinity is high in relation to (nonionic) surfactant-surface
67
affinity. Experiments were performed in sequential and competitive adsorption modes,
enabling the adsorption kinetic patterns to be interpreted in a fashion revealing the
dominant mode of surfactant-mediated stabilization of protein in each case.
4.2 Material and Methods
4.2.1. Materials and sample preparation
The standard sensor chips used for monitoring adsorption kinetics are
obtained from MicroVacuum Inc (Budapest, Hungary). It is made by depositing a thin
layer of Si0.25Ti0.75O2 by a sol-gel process to a glass chip. An optical waveguide grating,
which is approximately 20 nm deep, sinusoidal, and has 2400 lines/mm, is embossed to
the surface of the chip [4]. The sensor chips were used without further modification and
were soaked in the desired buffer overnight to enable equilibration of the waveguide film
with the cover medium.
The proteins used were lyophilized chicken egg white lysozyme (Sigma
Aldrich, Saint Louis, MO) and a recombinant protein provided by Amgen. Lysozyme
was dissolved in Dulbecco’s phosphate buffered saline (1X PBS, Invitrogen, Carlsbad,
CA) at 0.5 mg/mL , which equals to 0.073mM and was filtered using a 0.2 um PVDF
filter (Millipore, Billerica, MA). The recombinant protein is approximately 18.8 kDa that
consists of 174 amino acids with 2 disulfide bonds. It was provided in 10 mM sodium
acetate, 5% sorbitol at pH 4.0 at 3 mg/mL and was diluted to 0.4 mg/mL, which equals to
0.073mM and filtered using a 0.2um PVDF filter before use. Both protein concentrations
were measured with a UV spectrophotometer (Cary 50, Varian, Inc.) using 0.66 mg/ mL
cm as extinction coefficient for lysozyme and 0.78 for the recombinant protein.
68
The surfactants studied were Poloxamer 188 (Pluronic ® F68, BASF,
obtained from Sigma Aldrich, Saint Louis, MO) polysorbate 80 (PS 80, Croda, East
Yorkshire, England) and polysorbate 20 (PS 20, Croda, East Yorkshire, England). Stock
solutions at high concentrations of the surfactants were made in 1X PBS and in 10 mM
sodium acetate, 5% sorbitol at pH 4.0 buffers and diluted to the respective buffers to have
a molar ratio of 1:10 and 1:100 with the proteins evaluated. Buffer salts and sorbitol were
from obtained from JT Baker (Philipsburg, NJ).
To study the effect of salt, the recombinant protein was dialyzed in
10mM sodium acetate, 250mM sodium chloride, 5% sorbitol, at pH 4.0 using a Slide-Alyzer (10KDa MW cutoff, Thermo Fisher Scientific, Rockford, IL). Concentration was
measured with a UV spectrophotometer (Cary 50, Varian, Inc.) and diluted to 0.4mg/mL.
4.2.2. Evaluation of adsorption kinetics
The adsorption kinetics was recorded in-situ by Optical Waveguide
Lightmode Spectroscopy (OWLS, MicroVacuum Inc. Hungary). The basic principle of
operation is when linearly polarized HeNe (632.8 nm) laser is coupled by a diffraction
grating into a waveguide layer. At two distinct angles, light is propagated by total internal
reflection along the waveguide which is defined as incoupling. The properties of the
covering media determine the specific incident angle necessary for the light to propagate,
which is defined as the incoupling angle. From the angle and mathematical optics models,
the refractive index (𝑑𝐴 ) and thickness (𝑛𝐴 ) of the overlayer can be calculated. These can
then be used to estimate the immobilized mass (Γ) of the adsorbed overlayer [5]:
𝑑𝑛 −1
𝛤 = 𝑑𝐴 (𝑛𝐴 − 𝑛𝐶 ) � �
𝑑𝑐
69
where 𝐶 is the solution concentration of the protein, 𝑛𝐶 is the bulk solution refractive
𝑑
index, and 𝑑𝑛 is the refractive index increment and is usually 0.182 cm3/g for proteins in
𝑐
solution which was used consistently throughout all measurements.
The sensor chips that were soaked in the desired buffer overnight are
placed in a flow cell. After aligning the sensor chip properly and securing it tightly to
prevent any buffer leaks, the syringe pump is turned on to flow the desired buffer of
interest through the system at 100 µL/min. After establishing a stable baseline, 100 µL of
protein alone, surfactant alone, or protein+ surfactant mixture, which will be described in
detail in the following subsections, was injected using a Hamilton syringe via valve
diverter while the syringe pump was turned off. Adsorption was monitored for 30
minutes. The sample was then rinsed with the same buffer at 100 µL /min for 15 minutes.
4.2.2.1. Surfactant+protein co-adsorption
Protein concentration was kept constant and the amount of surfactant was
varied to obtain a protein to surfactant molar ratio of 1:10 and 1:100. Final concentration
of poloxamer (Pluronic® F68) was at 600ppm and 6000ppm and polysorbate 80 (PS 80)
and 20 (PS20) were studied at 90ppm and 900ppm. After stable baselines were achieved,
the protein and surfactant mixtures, which were incubated for at least 1 hour, were
introduced and monitored for 30 minutes followed by a rinsing step just by buffer for 15
minutes.
4.2.2.2. Surfactant+protein sequential adsorption
After running buffer achieved stable baseline signals, first Pluronic® F68
at 600 ppm was introduced and monitored for 30 minutes followed by a buffer rinsing
step for 15 minutes. A rinsing step was added to remove any surfactant in the bulk or
70
reversibly bound surfactant to the sensor. Then, the protein of interest was added and
monitored for 30 minutes followed by a buffer rinsing step for another 15 minutes.
4.2.2.3. Surfactant+protein co-adsorption pre-coated with surfactant
After buffer stabilization was achieved, 90ppm PS 80 or PS 20 was
introduced and monitored for 30 minutes. To maintain same concentration of surfactant
throughout the experiment, no rinse step was involved. After 30 minutes, protein
mixtures with 90ppm PS 80 or PS 20 was introduced and monitored for another 30
minutes followed by a buffer rinse for 15 minutes.
4.3 Results and Discussion
4.3.1. Surfactant+lysozyme co-adsorption
The adsorption kinetics recorded for surfactant solutions in the absence
of protein are presented in Figure 4.1. Both PS 80 and PS 20 displayed higher affinity for
the surface than Pluronic® F68 at each concentration tested, with each surfactant
adsorbing in a fashion not entirely elutable by surfactant-free buffer. The adsorption
kinetics of lysozyme in PBS in the absence and presence of PS 80 and PS 20 are shown
in Figure 4.2. While the presence of surfactant altered the adsorption kinetics to some
extent, upon elution the kinetic patterns were highly similar to that recorded for lysozyme
in the absence of surfactant, in each case. Joshi et al. found polysorbate 80 to have no
measurable effect on the adsorption of a human recombinant Factor VIII at a hydrophilic
silica surface [6]. While each polysorbate showed some affinity for the surface used in
the present experiments, it is possible that these adsorbed layers are dominated by
lysozyme after elution.
71
Adsorbed amount (ng/cm2)
200
90 ppm PS 80
900 ppm PS 80
90 ppm PS 20
900 ppm PS 20
600 ppm F68
6000 ppm F68
150
100
50
0
0
10
20
30
40
50
Time (min)
Figure 4.1: Adsorption kinetics of 90 and 900 ppm PS 80, 90 and 900 ppm PS 20, and
600 and 6000 ppm Pluronic® F68 in PBS
72
200
Adsorbed amount(ng/cm2)
0 ppm
900 ppm PS 80
900 ppm PS 20
150
100
50
0
0
10
20
30
40
50
Time (min)
Figure 4.2: Adsorption kinetics of lysozyme in PBS in the presence of 0 ppm, 900 ppm
PS 80 and 900 ppm PS 20
The adsorption kinetics of lysozyme in PBS in the presence of 0 ppm,
600ppm and 6000ppm Pluronic® F68 are shown in Figure 4.3. In contrast to the patterns
observed with the polysorbates, the amount of adsorption after the elution step is reduced
appreciably in the presence of F68, to a level near that expected for F68 in the absence of
protein. This could be due to F68 locating at the interface more rapidly than lysozyme,
thus inhibiting lysozyme adsorption. On the other hand, the F68 triblocks may have
associated with lysozyme in solution to form complexes of low adsorption affinity. As
shown in Figure 4.1 however, F68 itself shows very low affinity for the surface, and these
results likely suggest that the reduction in protein adsorption is owing to complex
formation and not to F68 location at the surface. This can be tested by the sequential
73
introduction of F68 and lysozyme to the surface.
200
Adsorbed amount (ng/cm2)
0 ppm
600 ppm F68
6000 ppm F68
150
100
50
0
0
10
20
30
40
50
Time (min)
Figure 4.3: Adsorption kinetics of lysozyme in PBS in the presence of 0 ppm, 600 ppm,
and 6000 ppm Pluronic® F68
4.3.2. Pluronic® F68 + lysozyme sequential adsorption
Figure 4.4 shows results of a sequential adsorption experiment in which
introduction of F68 to the surface is followed by introduction of lysozyme. This
intermediate rinse step at 30 min removes any loosely bound F68 at the interface as well
as any F68 present in solution. (Data recorded for lysozyme with no F68, from Figure 4.2,
was superimposed on Figure 4.4 beginning at 45 min for comparison.) As shown clearly
in Figure 4.4, the presence of surface-bound F68 did not prevent lysozyme adsorption.
Upon addition of lysozyme at 45 min, the adsorption kinetics were somewhat slower than
that recorded without the F68 “pre-coated” on the surface, possibly owing to
74
displacement of adsorbed F68 by lysozyme. The elution patterns are highly similar in
each case. These observations strongly support the thought that F68 does not inhibit
lysozyme adsorption by its preferential location to the interface. Rather, F68 inhibits
lysozyme adsorption by formation of less surface active, protein-surfactant complexes.
200
0 ppm
600 ppm F68
Adsorbed amount (ng/cm2)
Lysozyme
150
100
Rinse
Rinse
50
0
0
20
40
60
80
100
Time (min)
Figure 4.4: Adsorption kinetics of 600 ppm Pluronic® F68 followed by buffer elution
and the introduction of lysozyme in PBS.
4.3.3. Polysorbates +lysozyme co-adsorption pre-coated with polysorbates
Figure 4.5 shows polysorbate adsorption followed by the addition of
lysozyme + polysorbate, followed by elution with PBS buffer. (Data recorded for
lysozyme with no polysorbate, from Figure 4.2, was superimposed on Figure 4.5
beginning at 30 min for comparison.) PS 80 and PS 20 displayed appreciable adsorption
upon introduction, and were not eluted. Upon introduction of protein in the presence of
75
the PS 80, the adsorption kinetics were somewhat slower than that recorded without PS
80 “pre-coated” on the surface, likely owing to displacement of adsorbed PS 80 by
lysozyme. No appreciable change in kinetics was observed in the case of PS 20 +
lysozyme in relation to lysozyme alone, consistent with PS 80 showing a higher surface
affinity than PS 20. Finally, as in Figure 4.2, upon elution the kinetic patterns were highly
similar to that recorded for lysozyme in the absence of surfactant, in each case.
200
0 ppm
90 ppm PS80
90 ppm PS 20
Adsorbed amount (ng/cm2)
Lysozyme + polysorbates
150
100
Rinse
50
0
0
20
40
60
80
Time(min)
Figure 4.5: 90 ppm polysorbates adsorption followed by 90 ppm polysorbates +
lysozyme co-adsorption in PBS
4.3.4. Surfactants+ recombinant protein co-adsorption
The adsorption kinetics recorded for only surfactant solutions in 10 mM
sodium acetate, 5% sorbitol, pH 4.0 are presented in Figure 4.6. Both PS 80 and PS 20
displayed higher affinity for the surface than Pluronic® F68 at each concentration tested,
76
with each surfactant adsorbing in a fashion not entirely elutable by surfactant-free buffer.
Also, all of the surfactants displayed higher affinity in 10 mM sodium acetate, 5%
sorbitol, pH 4.0 compared to when in PBS (Figure 4.1). The adsorption kinetics of
recombinant protein in 10 mM sodium acetate, 5% sorbitol, pH 4.0 in the absence and
presence of PS 80, PS 20 and F68 are shown in Figure 4.7. Only the highest surfactant
concentrations are shown since the lower surfactant concentrations displayed similar
adsorption and elution behavior. All of the surfactants are seen to have a slight effect in
reducing the amount of protein on the surface upon rinsing. However, differences in the
elution patterns are observed when comparing lysozyme to recombinant protein in the
presence of surfactants. In the presence of F68, the amount of adsorption of the
recombinant protein after the elution step did not decrease significantly as observed with
lysozyme. However, in the presence of polysorbates, more effect was seen in the elution
step for the recombinant protein than for lysozyme which had almost none.
In a previous study, recombinant protein is shown to display more
surface activity than lysozyme at the air-water interface. Consequently, more adsorption
and less rinsing of the recombinant protein is seen when compared to lysozyme at the
same surface. The effect of surfactants on the adsorption kinetics could be due to the
recombinant protein being more surface active than lysozyme. Also, the surfactants,
especially the polysorbates showed higher affinity in 10 mM sodium acetate, 5% sorbitol,
at pH 4.0 than in PBS. This could be another factor for differences shown for the
recombinant protein.
77
250
90 ppm PS 80
900 ppm PS 80
90 ppm PS 20
900 ppm PS 20
600 ppm F68
6000 ppm F68
Adsorbed amount (ng/cm2)
200
150
100
50
0
0
10
20
30
40
50
Time (min)
Figure 4.6: Adsorption kinetics of 90 and 900 ppm PS 80, 90 and 900 ppm PS 20, and
600 and 6000ppm Pluronic® F68 in 10 mM sodium acetate, 5 % sorbitol, at pH 4.0
78
250
Adsorbed amount (ng/cm2)
200
150
100
0 ppm
6000 ppm F68
900 ppm PS 80
900 ppm PS 20
50
0
0
10
20
30
40
50
Time (min)
Figure 4.7: Adsorption kinetics of recombinant protein in 10 mM sodium acetate, 5%
sorbitol, pH 4.0 in in the presence of 0 ppm, 900 ppm PS 80, 900 ppm PS 20, and
6000ppm Pluronic® F68
4.3.5. Polysorbates+ recombinant protein co-adsorption pre-coated with polysorbates
Figure 4.8 shows polysorbate adsorption followed by the addition of
recombinant protein + polysorbate, followed by elution with 10 mM sodium acetate, 5%
sorbitol, pH 4.0 buffer. (Data recorded for recombinant with no polysorbate, from Figure
4.7, was superimposed on Figure 4.8 beginning at 30 min for comparison.) PS 80 and PS
20 displayed appreciable adsorption upon introduction, and were not eluted. Upon
introduction of protein in the presence of the PS 80, the adsorption kinetics were
somewhat slower than that recorded without PS 80 “pre-coated” on the surface, likely
owing to displacement of adsorbed PS 80 by recombinant protein. No appreciable change
in kinetics was observed in the case of PS 20 + recombinant protein in relation to
79
recombinant protein alone, consistent with PS 80 showing a higher surface affinity than
PS 20. Due to the higher affinity of the polysorbates at the surface, the amount of
adsorption after the elution step is reduced in the presence of polysorbates, more
significant for PS 80 than for PS 20.
250
Recombinant protein +Polysorbates
Adsorbed amount (ng/cm2)
200
150
Rinse
100
50
0 ppm
90 ppm PS 80
90 ppm PS 20
0
0
20
40
60
Time (min)
Figure 4.8: 90 ppm polysorbates adsorption followed by 90 ppm polysorbates +
recombinant protein co-adsorption in 10 mM sodium acetate, 5 % sorbitol, pH 4.0
4.3.6. Effect of salt on Pluronic® F68 +recombinant protein co-adsorption and
sequential adsorption
Our previous paper has shown that a mechanism for surfactant
adsorption may be governed by the formation of a rate-limiting structural intermediate
(i.e., an “activated complex”) between surfactant aggregates and protein [3]. While
Pluronic® F68 is not expected to form micelles in water except at very high
80
80
concentrations[7-10]; it is known to exhibit greater aggregation and micellization in salt
solutions[11]. Since Pluronic® F68 was not as effective in reducing the recombinant
protein adsorption compared to lysozyme; addition of 250 mM in 10 mM sodium acetate,
5 % sorbitol, at pH 4.0 was studied to verify the role of surfactant aggregates.
As shown in Figure 4.9, the amount of recombinant protein adsorbed
reduced when the ionic strength was increased. The presence of salt at higher
concentrations effectively shields the charges on the surface and on the protein which is
well documented. The adsorption kinetics of recombinant protein in 10 mM sodium
acetate, 5% sorbitol, 250 mM sodium chloride, at pH 4.0 in the presence of 0 ppm,
600ppm and 6000ppm Pluronic® F68 are shown in Figure 4.10. Reduction of
recombinant protein adsorption with Pluronic® F68 in the elution step is more significant
in the presence of salt than without salt as shown in Figure 4.7. The addition of 250 mM
sodium chloride may have induced Pluronic® F68 aggregation which associated with
recombinant protein more, thus forming lower surface affinity complexes. As a result,
less adsorption is observed for recombinant protein in the presence of Pluronic® F68 in
250mM sodium chloride.
Figure 4.11 shows results of a sequential adsorption experiment in which
introduction of F68 to the surface is followed by introduction of recombinant protein.
This intermediate rinse step at 30 min removes any loosely bound F68 at the interface as
well as any F68 present in solution. (Data recorded for recombinant protein with no F68,
from Figure 4.10, was superimposed on Figure 4.11 beginning at 45 min for comparison.)
As shown clearly in Figure 4.11, the presence of surface-bound F68 did not prevent
recombinant protein adsorption. Upon addition of recombinant protein at 45 min, the
adsorption kinetics were similar with that recorded without the F68 “pre-coated” on the
surface. The elution patterns are highly similar in each case. These observations strongly
81
support the thought that F68 does not inhibit recombinant protein adsorption by its
preferential location to the interface. Rather, in the presence of salt, F68 inhibits
recombinant protein by formation of less surface active, protein-surfactant complexes.
250
Adsorbed amount (ng/cm2)
200
150
100
50
0 mM NaCl
250 mM NaCl
0
0
10
20
30
40
50
Time (min)
Figure 4.9: Effect of salt concentration on the adsorption of recombinant protein in 10
mM sodium acetate, 5 % sorbitol, at pH 4.0
82
250
0 ppm + 250 mM NaCl
600 ppm F68 + 250 mM NaCl
6000 ppm F68 + 250 mM NaCl
Adsorbed amount (ng/cm2)
200
150
100
50
0
0
10
20
30
40
50
Time (min)
Figure 4.10: Adsorption kinetics of recombinant protein in 10 mM sodium acetate, 5 %
sorbitol, 250 mM sodium chloride, at pH 4.0 in the presence of 0 ppm, 600ppm, and
6000ppm Pluronic® F68
83
250
Rinse
Recombinant protein
Adsorbed amount (ng/cm2)
200
150
100
Rinse
50
0 ppm + 250 mM NaCl
600 ppm F68 + 250 mM NaCl
0
0
20
40
60
80
100
Time (min)
Figure 4.11: Adsorption kinetics of 600ppm Pluronic® F68 followed by buffer elution
and the introduction of recombinant protein in 10 mM sodium acetate, 5 % sorbitol, 250
mM sodium chloride, at pH 4.0.
4.4 Conclusions
To determine the dominant mode of surfactant-mediated stabilization of
protein, lysozyme and a more surface active recombinant protein was studied in the
presence of Pluronic® F68, PS 80 and PS 20 at solid surfaces. Pluronic® F68 was
effective in reducing protein adsorption especially in the presence of salt by forming
lower surface affinity complexes. PS 80 and PS 20 were not effective in preventing
lysozyme adsorption and only slight reductions were observed for the recombinant
protein adsorption. Even though polysorbates were observed to adsorb at the surface,
protein was able to replace all or most of the adsorbed surfactant owing to the weak
polysorbate-surface interaction.
84
4.5 References
[1] J. Carpenter, B. Cherney, A. Lubinecki, S. Ma, E. Marszal, A. Mire-Sluis, T.
Nikolai, J. Novak, J. Ragheb, and J. Simak, Meeting report on protein particles and
immunogenicity of therapeutic proteins: filling in the gaps in risk evaluation and
mitigation. Biologicals 38 (2010) 602-11.
[2] J.S. Bee, T.W. Randolph, J.F. Carpenter, S.M. Bishop, and M.N. Dimitrova,
Effects of surfaces and leachables on the stability of biopharmaceuticals. J Pharm Sci
(2011).
[3] H.J. Lee, A. McAuley, K.F. Schilke, and J. McGuire, Molecular origins of
surfactant-mediated stabilization of protein drugs. Adv Drug Deliv Rev 63 (2011) 116071.
[4] A. Szekacs, N. Adanyi, I. Szekacs, K. Majer-Baranyi, and I. Szendro, Optical
waveguide light-mode spectroscopy immunosensors for environmental monitoring. Appl
Opt 48 (2009) B151-8.
[5] J.A. De Feijter, J. Benjamins, and F.A. Veer, Ellipsometry as a tool to study
the adsorption behavior of synthetic and biopolymers at the air–water interface.
Biopolymers 17 (1978) 1759-1772.
[6] O. Joshi, J. McGuire, and D.Q. Wang, Adsorption and function of
recombinant factor VIII at solid–water interfaces in the presence of Tween-80. J Pharm
Sci 97 (2008) 4741-4755.
[7] P. Bahadur, P. Li, M. Almgren, and W. Brown, Effect of potassium fluoride
on the micellar behavior of Pluronic F-68 in aqueous solution. Langmuir 8 (1992) 19031907.
[8] H.-W. Tsui, Y.-H. Hsu, J.-H. Wang, and L.-J. Chen, Novel Behavior of Heat
of Micellization of Pluronics F68 and F88 in Aqueous Solutions. Langmuir 24 (2008)
13858-13862.
[9] Y. Li, M. Bao, Z. Wang, H. Zhang, and G. Xu, Aggregation behavior and
complex structure between triblock copolymer and anionic surfactants. Journal of
Molecular Structure 985 (2011) 391-396.
[10] S. Borbely, and J.S. Pedersen, Temperature-induced aggregation in aqueous
solutions of pluronic F68 triblock copolymer containing small amount of o-xylene.
Physica B: Condensed Matter 276-278 (2000) 363-364.
[11] K. Patel, B. Bharatiya, Y. Kadam, and P. Bahadur, Micellization and
Clouding Behavior of EO–PO Block Copolymer in Aqueous Salt Solutions. Journal of
Surfactants and Detergents 13 89-95.
85
CHAPTER 5
GENERAL CONCLUSION
A simple thermodynamic analysis indicated the presence of protein
caused a reduction in ∆𝑮 for the surfactant adsorption process, with this reduction
deriving entirely from a reduction in ∆𝑯. We suggest that protein accelerates the
adsorption of these surfactants by disrupting their self associations, releasing surfactant
monomers. The increased concentration of surfactant monomers may promote surfactant
adsorption, or the formation of stable, surfactant-protein complexes having little or no
effect on surfactant adsorption rate. Based on this, we expect that accelerated surfactant
adsorption in the presence of protein will occur with surfactants that stabilize protein
mainly by their own adsorption at interfaces, and we expect the absence of accelerated
surfactant adsorption will occur with surfactants that form stable surfactant-protein
associations. Pluronic® F68 was shown to be effective in reducing protein adsorption
especially in the presence of salt by forming lower surface affinity complexes. PS 80 and
PS 20 were not effective in preventing lysozyme adsorption and only slight reductions
were observed for the recombinant protein adsorption. Even though polysorbates were
observed to adsorb at the surface, the protein was able to replace all or most of the
adsorbed surfactant owing to the weak polysorbate-surface interaction. In summary PS 20
and PS 80 are able to inhibit protein adsorption only by their preferential location at the
interface, and not by formation of less surface active, protein-surfactant complexes. On
the other hand, F68 clearly is able to inhibit protein adsorption by formation of proteinsurfactant complexes, and not by its preferential location at the interface.
86
Chapters 2-4 present how surfactants interact with interfaces and proteins
(particularly the preferential location of a surfactant at an interface, or its association with
protein in solution), and can provide guidance in selecting surfactants and excipients to
reduce protein losses in a given application.
87
BIBLIOGRAPHY
A. Gigout, M.D. Buschmann, M. Jolicoeur, The fate of Pluronic® F-68 in
chondrocytes and CHO cells, Biotechnol. Bioeng. 100 (2008) 975-987.
A. Szekacs, N. Adanyi, I. Szekacs, K. Majer-Baranyi, and I. Szendro, Optical
waveguide light-mode spectroscopy immunosensors for environmental monitoring. Appl
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