REVIEWS

Effects of Surfaces and Leachables on the Stability

of Biopharmaceuticals
JARED S. BEE,1 THEODORE W. RANDOLPH,2 JOHN F. CARPENTER,3 STEVEN M. BISHOP,1 MARIANA N. DIMITROVA1
1
Formulation Sciences Department, MedImmune, Gaithersburg, Maryland 20878
2
Department of Chemical and Biological Engineering, University of Colorado, Boulder, Colorado 80309
3
Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, Aurora, Colorado 80045

Received 19 October 2010; revised 11 January 2011; accepted 12 April 2011

Published online 26 April 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22597

ABSTRACT: Therapeutic proteins are exposed to various potential contact surfaces, particles,
and leachables during manufacturing, shipping, storage, and delivery. In this review, we present
published examples of interfacial- or leachable-induced aggregation or particle formation, and
discuss the mitigation strategies that were successfully utilized. Adsorption to interfaces or
interactions with leachables and/or particles in some cases has been reported to cause protein
aggregation or particle formation. Identification of the cause(s) of particle formation involving
minute amounts of protein over extended periods of time can be challenging. Various formulation
strategies such as addition of a nonionic surfactant (e.g., polysorbate) have been demonstrated
to effectively mitigate adsorption-induced protein aggregation. However, not all stability prob-
lems associated with interfaces or leachables are best resolved by formulation optimization.
Detectable leachables do not necessarily have any adverse impact on the protein but control
of the leachable source is preferred when there is a concern. In other cases, preventing pro-
tein aggregation and particle formation may require manufacturing process and/or equipment
changes, use of compatible materials at contact interfaces, and so on. This review summarizes
approaches that have been used to minimize protein aggregation and particle formation dur-
ing manufacturing and fill–finish operations, product storage and transportation, and delivery
of protein therapeutics. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J
Pharm Sci 100:4158–4170, 2011
Keywords: protein aggregation; formulation; stability; agitation; air–water interface; adsorp-
tion; particles; leachables; surface; biopharmaceuticals characterization

INTRODUCTION formation, with the resulting degradation products

Therapeutic proteins are used to treat a wide range
of serious medical conditions, providing substantial
benefits to patients. Proteins are complex molecules,
subject to both intrinsic variation (e.g., glycosylation
pattern and charge isoforms) and a variety of chemi-
cal (e.g., deamidation and oxidation) and physical (for-
mation of soluble aggregates, particle formation, and
reversible association) degradation pathways. Most
common intrinsic degradation pathways for protein
therapeutics include aggregation and often particle
Abbreviations used: mAb, monoclonal antibody.
Correspondence to: Jared S. Bee (Telephone: +301–398-5912;
Fax: +303-492-4341; E-mail: BeeJ@medimmune.com, jaredsbee
@gmail.com)
Journal of Pharmaceutical Sciences, Vol. 100, 4158–4170 (2011)
© 2011 Wiley-Liss, Inc. and the American Pharmacists Association
normally making up a very small mass fraction of the
therapeutic protein product. Not all molecular vari-
ants or degradation products necessarily result in a
loss of efficacy or a decrease in safety. Some types of
protein aggregates may elicit immune responses in
patients.1,2 However, the mechanisms for immuno-
genicity of therapeutic proteins in patients are still
not well understood and a link between immunogenic-
ity and aggregates or particles in products remains
unclear in many cases.3,4
Using state-of-the-art technology, biotechnology
companies use formulation and process control strate-
gies to obtain high purity and stability in order to
meet a typical goal of a 2-year shelf life.5 For the
general case of bulk protein aggregation as described
by Chi et al.,6 either partial unfolding or aggre-
gate assembly can be the rate-determining step for

4158 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
 SURFACES AND LEACHABLES EFFECTS ON BIOPHARMACEUTICALS 4159

aggregation of proteins. The conformational and col-
loidal stability of the protein can be optimized through
the appropriate use of formulation buffer type, pH,
and excipients.5–10 Similarly, formulation conditions
can also be used to maximize chemical stability of
proteins.5–10 High-concentration monoclonal antibody
(mAb) products present their own unique challenges
such as self-association, viscosity, opalescence, and
protein particle formation.11,12
Protein stability in bulk solution is only one of
the key issues. During manufacturing, final fill–fin-
ish, storage, and delivery, proteins may adsorb to sur-
faces or react/bind with leachables. In some cases,
this has resulted in aggregation, particle formation,
or adsorption losses.5,10 Figure 1 depicts some of the
processes of how solid and liquid contact surfaces and
leachables have caused instabilities in protein prod-
ucts. Adsorption of proteins to surfaces is a complex
process that is important in many fields.13,14 Protein
surface adsorption can be driven by a combination
of electrostatic forces, hydrophobic binding interac-
tions, and entropy changes due to contributions from
both water and protein.14,15 These surface adsorption
processes may be reversible or irreversible and may
lead to either unfolding or partial unfolding of the ad-
sorbed protein or only minimal perturbations to the
protein structure. Depending on these factors, the ad-
sorption of protein may be minimal and not cause any
additional aggregation or particle formation. Simple
adsorption can result in a reduction in the bulk pro-
tein concentration that can be more of a concern for
low concentration formulations. In other cases, pro-
tein adsorption could nucleate further aggregation
and particle formation. If adsorption is reversible,
it is possible that the desorbed proteins may be re-
leased in a structurally perturbed form that could
lead to further aggregation or particle formation in
the bulk.16 However, the detailed mechanism(s) has

N P
A –

P I
N
V

P S
P

M
L

H
C

A A

Figure 1. Possible physical degradation pathways of proteins caused by interfaces, foreign

particulates, and leachables described in this review. The processes in the figure correspond to
specific examples that have been published and are discussed in the text. Although the figure
shows a vial as one example, these processes may also occur in other upstream operations and
in other containers or delivery devices. These examples are also described and reviewed in this
work.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
4160 BEE ET AL.
not been fully determined for many published reports
of adverse protein interactions with surfaces. Figure 1
illustrates alternative mechanisms of particle forma-
tion. These include agglomeration of protein-coated
particles or silicone oil droplets and coagulation of
proteins with leachables. This might occur when a
few subvisible particles (SVPs) that were initially col-
loidally stable (due to a high negative surface charge
in the case of glass and silicone oil at common formu-
lation pH conditions) become less stable when the sur-
face charge is reduced by adsorption of protein. These
protein-coated particles may then simply agglomerate
together to form larger, more easily detectable parti-
cles. It is also possible that foreign particles could ag-
glomerate even if there is little or no protein adsorp-
tion to the particles. A similar process of binding of
leachables to proteins can lead to particle formation
through colloidal destabilization of the protein, fol-
lowed by precipitation of particles. Other leachables
may also cause protein damage by directly reacting
with the protein, potentially creating an aggregation-
competent protein species. The last process for ag-
gregation and particle formation we discuss is expo-
sure to the air–water interface. Air–water interface
exposure is one of the more common causes of par-
ticle formation and aggregation described in the lit-
erature. As with other interfaces, the details of the
mechanism(s) of air–water interface-induced aggre-
gation are not well described for many proteins. In
this review, we present examples of the published ev-
idence for these aggregation and particle formation
processes and discuss rational mitigation strategies.
Many of these examples of interface- and leachable-
induced aggregation and particle formation processes
are specific to certain products or conditions. We also
note that detectable leachables may have no adverse
impact of product safety, efficacy, quality, or protein
stability. In this review, we have included many dif-
ferent examples (even if they are less common) so that
the lessons learned may be used to help in the practi-
cal resolution of other similar issues in the future.
MANUFACTURING AND FILL–FINISH
OPERATIONS
Manufacturing of therapeutic proteins is a complex
process, which begins with production of the protein
in cells cultured in a bioreactor wherein the protein
is exposed to a multitude of solution species in the
growth medium. The protein is then separated from
the cell culture media by filtration or centrifugation.
Recovery from inclusion bodies and refolding are per-
formed if necessary. In downstream protein purifica-
tion, viral inactivation and removal steps are often
performed (e.g., low pH incubation, nanofiltration,
and solvent–detergent addition). Multiple chromatog-
raphy (e.g., affinity, ion exchange, and hydrophobic
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
interaction) and filtration steps are used to purify
the protein further. The protein may be concentrated
and formulated using diafiltration. The formulated
bulk may then be frozen or held before the final
sterile filtration and fill–finish operations. Following
the final sterile filtration step, the product is filled
into vials, syringes, or cartridges. Each of these steps
may expose the protein to interfaces (i.e., solid–liq-
uid and air–liquid) under a variety of solution condi-
tions. In this review, we focus on downstream exam-
ples of interfacial protein instabilities, although many
of the aggregation and particle formation processes to
which proteins are exposed during downstream unit
operations could also be relevant to the cell culture
environment.
Diafiltration
Air bubble entrainment and/or microcavitation have
been cited as a cause of aggregation during diafil-
tration operations.17–19 Adsorption to solid surfaces,
contamination by particulates, and increased rate of
aggregate assembly due to mixing could also be causes
of aggregation.19 Simple adsorption losses and fouling
of the protein onto the membrane can also occur. For
instance, deactivation of aminoacylase was directly
caused by adsorption losses to an ultrafiltration mem-
brane surface.20 The type and brand of filtration mem-
brane have been shown to result in different levels of
protein adsorption.21
Process controls may be used to minimize aggrega-
tion during diafiltration by optimization of the oper-
ation parameters such as the transmembrane pres-
sure and cross-flow rate.22 It has been suggested
that reducing turnover of the air–water interface and
bubble entrainment would also reduce the formation
of particles in biotherapeutics during diafiltration
operations.18,19 It is possible that some formulation
excipients can provide additional protection during
diafiltration. This of course depends upon whether ex-
cipients are added during the diafiltration operation
or afterward by addition of a concentrated stock of
the excipients. Although addition of a surfactant can
suppress the formation of aggregates at the air–water
interface when the protein is also exposed to shear,23
this strategy may not be practical for diafiltration op-
erations. Surfactants are normally added after the
diafiltration operation because of the difficulties in
controlling and predicting the final surfactant level
in the retentate.24
Freezing and Thawing
Freezing is a common unit operation during the pro-
duction of therapeutic protein products. Bulk inter-
mediates are often frozen to increase their stability
during production hold steps and freezing is the first
step in lyophilization. Freezing and thawing can trig-
ger aggregation and particle formation in proteins by
DOI 10.1002/jps
 SURFACES AND LEACHABLES EFFECTS ON BIOPHARMACEUTICALS
various different mechanisms.25 Storage temperature
and freezing rate are important parameters for frozen
stability. One other factor that is the focus of this re-
view contributing to the overall destabilization of the
protein is the choice of container material. For ex-
ample, polytetrafluoroethylene and other commercial
freezing containers fostered more aggregation than
polypropylene during freeze–thawing of an IgG2 .26
Cryoprotectants, such as sucrose or trehalose, are of-
ten added to protein formulations to protect against
freezing and thawing damage.25
The ice–solution interface itself can be destabi-
lizing to proteins: increased intermolecular $-sheet
content was measured by infrared spectroscopy for
two different proteins adsorbed to the ice surface.27
Polysorbate addition has been shown to reduce for-
mation of nonnative intermolecular $-sheet levels in
proteins adsorbed to ice interfaces.27 In this case,
the ability of polysorbate to reduce such structural
changes in ice-adsorbed protein molecules was pro-
tein specific.27 Polysorbate 20 protected Factor XIII
during freeze–thawing by competing with the par-
tially unfolded protein for interfaces.28 Additionally,
polysorbate 80 protected hemoglobin from damage
at interfaces during freezing.29 The 40% or greater
loss of interleukin-11 (IL-11) activity caused by ad-
sorption to glass lyophilization vials was prevented
by polysorbate 20, although for complete protection
during lyophilization, trehalose and human albumin
were also necessary.30
The rate of cooling and the degree of supercooling
affect the number and sizes of ice crystals and the
time the protein is exposed to the ice interface. Each
of these variables could potentially influence the ex-
tent of freeze–thawing-induced protein aggregation.
Because there are multiple variables in freeze–thaw
stress, experimental studies to test the sensitivity of
the specific protein formulation to realistic and worst-
case freeze–thaw stresses can be used to determine
appropriate mitigation strategies.
Sterile Filtration and Fill–Finish
Sterile filtration and fill–finish operations may exert
adverse effects on stability by exposing the protein to
production equipment surfaces (e.g., those presented
by membranes, tubing, and pumps). In an engineering
approach, choice of equipment to minimize air–water
interface exposure and turnover, particle shedding,
leachables, and cavitation can be employed to elimi-
nate or minimize suspected causes of aggregation.19
This type of optimization should be performed while
also maintaining product homogeneity (i.e., ensuring
mixing is adequate) and sterility, and overall robust-
ness and quality. These same strategies could also
be useful to minimize aggregation or particle forma-
tion in other upstream unit operations. Formulation
approaches can also be very effective at reducing ad-
DOI 10.1002/jps
4161
verse interactions with interfaces. For instance, the
aggregation leading to membrane fouling during ster-
ile filtration of human growth hormone was found to
be caused by adsorption to hydrophobic interfaces and
could be mitigated by addition of surfactant.31 Differ-
ences in the magnitude of protein adsorption has been
observed between different types and brands of ster-
ilizing filters (e.g., polyvinylidene fluoride (PVDF),
polyethersulfone (PES), cellulose acetate (CA), and
Nylon).21 Various filters were found to adsorb polysor-
bate 80, requiring appropriate setup of the preflush
step to avoid decreasing the levels of surfactant below
the intended value for the final protein formulation.21
Interestingly, it has been found that cellulose could
preferentially adsorb soluble aggregates of a mAb
from solution, although this did not have any adverse
effect on the protein stability in bulk solution.32
Stainless steel is ubiquitous in protein produc-
tion equipment and has been reported to be a cause
of protein aggregation or fragmentation in several
cases: submicron steel particles shed from a pump in
the laboratory environment caused “agglomeration of
protein-coated particles” (see Fig. 1) and/or nucleated
formation of larger aggregates of a mAb33 ; Fe ions
caused hinge-region fragmentation of a mAb34 ; expo-
sure to the stainless steel surface combined with ad-
ditional shear stress resulted in aggregation of a mAb
that exhibited a first-order dependence on protein
concentration35 ; Fe ions leached from steel caused
oxidation and aggregation36 ; Fe ions directly bound
to a protein resulting in conformational destabiliza-
tion followed by aggregation,37 and surface-induced
soluble aggregation of a mAb had a second-order de-
pendence on steel surface area and a zero-order de-
pendence on bulk protein concentration that could
be completely suppressed by polysorbate.38 Stainless
steel surfaces typically are “passivated” or “electropol-
ished” to create a more corrosion-resistant chromium
oxide-rich surface layer. Factors that may impact the
protein in solution include the following: the grade of
steel alloy, the frequency of passivation, and chemical
exposures of the steel. The impact of the formulation
may play a particularly large role in the potential ad-
verse interactions; for instance, exposure of steel to
chloride ions at low pH has been shown to result in
corrosion and release of Fe ions that subsequently cat-
alyzed the oxidation of methionine residues.36 Stain-
less steel exposure is an example of where there may
be multiple distinct causes of aggregation or par-
ticle formation: the steel surface itself, steel parti-
cles shed from equipment, and the Fe ions leached
from steel equipment. These examples would corre-
spond to the scenarios of “physical or chemical in-
stability caused by leachables” (Fe ions), “nucleation
of aggregates on heterogenous particles or surfaces”
(steel surface), and “agglomeration of protein-coated
particles” (steel particles) shown in Figure 1. Here,
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
4162 BEE ET AL.
correct identification of the cause of the aggregation,
fragmentation, or particle formation is crucial for cre-
ating an effective mitigation strategy. Addition of a
surfactant would be expected to reduce aggregation
induced by surface adsorption, yet may not be effec-
tive in eliminating oxidation, fragmentation, or con-
formational destabilization caused by Fe ions. Rather,
direct reduction of Fe ion levels by frequent passiva-
tion of equipment and avoiding exposures of steel to
extreme low pH in the presence of chloride or other
corrosive ions might be a better strategy to eliminate
negative effects of Fe ions on protein stability.36 Ad-
dition of metal chelators has also been shown to be
effective in eliminating the multiple adverse effects
of Fe ions on protein stability, although care must be
taken in the choice and level of the chelator.37 Nu-
cleation of larger visible particles from smaller steel
particles shed from pumps may not be completely sup-
pressed by surfactant.26 In this scenario, a change in
the process equipment has been shown to be effective.
For instance, protein particle formation during fill-
ing of an IgG was eliminated by replacement of a ra-
dial piston pump with a rolling diaphragm pump.39 In
some cases, there may be synergistic or compounded
effects that may make identification of the problem
and correct mitigation more difficult. A good example
is where buffer-dependent conformational changes in
a mAb increased the exposure of a site sensitive to
Fe-catalyzed fragmentation.34
Stainless steel is not the only important in-process
surface to consider. In recent years, use of dis-
posable containers has become a common practice
in various steps of the manufacturing of protein
therapeutics. Disposable containers pose potential
challenges associated with leachables and possible
shedding of particles, and are usually subjected to ex-
tensive evaluation by biopharmaceutical companies
before implementation.40
CONTAINER CLOSURE
Glass vials with rubber stoppers made of various poly-
mers and coatings are commonly used primary con-
tainers for protein therapeutics. Most recently, vials
or syringes made of cyclic polyolefin (clear plastic) are
being evaluated as options for container-closure ma-
terials for some biopharmaceuticals.41 Container clo-
sures can be exposed to various solvents to extract and
identify compounds that are then monitored as leach-
ables under realistic product contact conditions.40
This can result in an identification of a large number
of extractables that are often not actually detectable
in the formulation upon extended product contact. Di-
rect health-based risk assessments can then be per-
formed based on the extractables–leachables data for
a given product configuration.40 Indirect effects of
leachables could potentially include aggregation or
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
particle formation.40 Detectable leachables may not
necessarily have any adverse effects on product safety,
efficacy, or quality.
Rapid growth in the applications of targeted
biotechnology products is driving the development of
alternative delivery systems including prefilled sy-
ringes (PFSs), autoinjectors (AIs), and infusion de-
vices. Multiple commercial products are currently of-
fered as PFSs and AI devices, and the number is
expected to rapidly grow. The development of PFSs,
AI, and infusion devices are associated with potential
for component compatibility challenges. These poten-
tial challenges include sensitivity of proteins to the
silicone oil often used to enhance the gliding perfor-
mance of the syringe/device, sensitivity to trace lev-
els of metals such as tungsten, which may be used in
the manufacturing of glass syringes with staked nee-
dles, and potential leachables from the glass, silicone,
rubber, and adhesive contact surfaces. These possible
adverse interactions are addressed during compati-
bility and stability studies during development. In
addition, various types of syringes are being devel-
oped currently by multiple vendors including silicone
oil- and tungsten-free syringes, enabling a greater se-
lection of container-closure systems to be potentially
chosen from and/or evaluated during development.
Glass
Borosilicate glass is the most commonly used primary
container material for biopharmaceuticals.41 During
development, each product formulation is generally
assessed and optimized for stability in glass vials
(with stopper). Glass vials surface properties can vary
between manufacturers and may change due to in-
teractions with the solution or sterilization proce-
dures, which could potentially result in pitting or
delamination.41–44 Glass has been successfully used
for many commercial protein products without caus-
ing aggregation or particle formation. Although re-
ports of glass delamination are extremely rare for
biotechnology products, the recent voluntary recall of
a commercial protein therapeutic because some lots
“. . .may contain extremely thin glass flakes (lamellae)
that are barely visible in most cases” shows that de-
lamination is still an important quality factor to be
considered.45 We note that the voluntary recall also
states that “To date, there have been no complaints
or adverse events reported which can be directly at-
tributed to the presence of glass lamellae.”45
Excipients can also potentially interact with leach-
ables from glass. Depending upon the exact supplier,
glass can potentially leach ions such as barium or
aluminum forming insoluble visible particles of bar-
ium sulfate or aluminum phosphate when exposed
to formulation excipients (sulfate and phosphate).46
Proteins can adsorb to glass surfaces. In one case,
the adsorption of protein to glass was minimized by
DOI 10.1002/jps
 SURFACES AND LEACHABLES EFFECTS ON BIOPHARMACEUTICALS
use of a siloxane coating and addition of surfactant.47
Although they do not have identical surface chem-
istry, silica nanoparticles have been used as a surro-
gate surface for glass and were found to nucleate the
growth of aggregates of recombinant human platelet-
activating factor acetylhydrolase.48 Adsorption of the
protein to glass and resulting aggregation could be
reduced by choosing a solution pH at which both the
protein and the glass had a net negative charge.48
Also, in a more recent study with a mAb, it was found
that there were differences in adsorption affinity as
a function of pH and a surprising tendency for pref-
erential adsorption of aggregates when the mAb was
formulated with 150 mM NaCl in 10 mM sodium ac-
etate at pH 5.0 and 5.5, and 150 mM NaCl in sodium
phosphate at pH 6.0 and 6.5.49 It has been shown that
for several mAbs, the structural changes on adsorp-
tion to spiked glass particles were minimal, however,
the protein-coated glass particles were less colloidally
stabile, resulting in formation of larger agglomerated
clusters of the spiked glass particles.32,50 This spe-
cific example corresponds to the general phenomenon
of “agglomeration of protein-coated particles” illus-
trated in Figure 1. The interactions between glass
and protein are clearly both protein specific and for-
mulation dependent. Simple adsorption losses at low
concentrations may be reduced by use of coatings or
surfactants.
Stoppers
Vial stoppers are often made from butyl and halobutyl
rubber that may also contain other additives or poten-
tial leachables depending upon the proprietary com-
pound formulation.41 Not all detectable leachables
will necessarily have any negative impact on product
quality. In one case, vial stoppers released metal ions
that in turn activated a metalloprotease, which then
resulted in protein product damage.1 Polytetrafluo-
rethylene (PTFE) or other proprietary polymers have
been used to coat vial stoppers to reduce the leaching
of rubber components into the bulk formulation. The
pure red cell aplasia (PRCA) experienced by Eprex
R
(Johnson & Johnson, New Brunswick, NJ) patients
has been attributed to leachables released from un-
coated rubber stoppers in the presence of polysorbate
used to replace human albumin as a stabilizer.51–53 In
other work, extractables from stoppers were not able
to elicit “danger signal” responses in dendritic cells
that would indicate their immunogenic potential.54
Association of protein with polysorbate micelles has
also been suggested as a possible cause of the im-
mune responses,55 although this particular hypothe-
sis has been questioned after additional studies by
other workers suggested that association with mi-
celles does not occur.53,56 The fundamental details and
cause(s) of the observed PRCA cases remain the sub-
DOI 10.1002/jps
4163
ject of ongoing research and debate. Ultimately, the
incidence of PRCA decreased when a FluroTec
R
(West
Pharmaceutical Services, Inc., Lionville, PA) stopper
configuration replaced the uncoated stopper and the
route of administration was changed from subcuta-
neous to intravenous.40,52
Container-closure changes are considered high
risk by regulatory agencies for parenteral biotech
products.1 Optimization of the formulation and
container-closure configuration is performed together.
Although use of a PTFE stopper apparently improved
the stability of Eprex
R
, PTFE was found to cause the
aggregation of insulin in accelerated studies.57 For-
mulation is important: Factor VIII adsorption to hy-
drophobic surfaces, with concomitant unfolding and
loss of activity of the adsorbed protein, could be re-
duced by polysorbate 80.58 Interestingly, adsorption
of Factor VIII to hydrophilic surfaces was not reduced
by polysorbate, although no associated unfolding or
loss of activity of the adsorbed protein was observed
as was seen for hydrophobic surfaces.58 Stoppers are
also often coated with silicone oil, and the amount of
silicone coated is one variable that can be controlled.
Vials are normally shipped and stored upright so the
exposure to the stopper might be minimal for an ac-
tual product. But for accelerated degradation studies,
incubation in vials that are inverted or laid side-on
can also be used to assess the impact of the stopper
on the stability of the product.
Tungsten and Needle Glue
Contamination of glass syringes with tungsten com-
pounds released from the tungsten pin used to form
the needle-mounting hole was identified as a cause of
protein particle formation,59 and has been reported
to have resulted in the formation of visible parti-
cles in at least two therapeutic protein products.1
In a recent study, it was demonstrated that tung-
sten metal particles dissolved into negatively charged
polyanions that electrostatically aggregated with a
mAb, resulting in rapid visible particle formation.60
Aggregation of the mAb by tungsten species was
only observed below about pH 6.0 and above tung-
sten levels of about 3–9 ppm, consistent with the
fact that tungsten polyanions are the major soluble
species formed under these conditions.60 The small
number of tungsten metal particles that need to dis-
solve to achieve this level of soluble polyanions high-
lights their potential potency in causing aggrega-
tion at lower pHs.60 Tungsten polyanion aggregation
with proteins was largely reversible in phosphate-
buffered saline.60 The mechanism of this aggrega-
tion of protein with tungsten polyanions can be
described as “coagulation:” coagulation is a colloidal
science term for when a colloid (in this case the pro-
tein) is electrostatically bridged and cross-linked by
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
4164 BEE ET AL.
charged species in solution (in this case tungsten
polyanions), leading to particles and precipitate for-
mation. Results for Fc fusion and "-helical proteins
spiked with extracts from actual tungsten pins used
in syringe-forming operations were also consistent
with polyanion-induced coagulation.61 Particle forma-
tion was greatest at lower pHs and higher soluble
tungsten levels, but in all cases, particles were only
formed at tungsten levels 10 times higher than those
actually measured in commercial PFS.61 These ex-
amples correspond to “coagulation with leachables”
as depicted in Figure 1. In response to the potential
tungsten-induced protein degradation, syringe man-
ufacturers have developed proprietary manufactur-
ing processes and engineering changes that control
or effectively eliminate residual tungsten contamina-
tion in glass-staked needle syringes. Here we have
referenced work showing different tungsten sensitiv-
ities of a mAb, an Fc fusion protein, and an "-helical
protein.60,61 These sensitivities varied based on mul-
tiple factors such as the protein-to-tungsten ratio, the
protein properties, protein concentration, and the for-
mulation conditions.60,61 Therefore, sensitivity of dif-
ferent proteins and different formulations to tungsten
leached from forming pins should be evaluated on a
case-by-case basis because there may, or may not, be
an impact at the actual potential worse case expected
tungsten levels in PFSs.60,61 It is likely that the re-
ductions of tungsten level in syringes, combined with
knowledge of the pH effects on potential coagulation
with protein, can now be used to effectively mitigate
this specific issue for proteins which are sensitive to
tungsten.
The US Food and Drug Administration (FDA) re-
ported in one case that if the specific glue used to
attach the needle to the syringe tip was not allowed
to fully dry, it could leach solvents, resulting in pro-
tein oxidation.46 They reported that in this specific
case, the issue could be resolved by allowing the glue
to fully dry for 6 months prior to using the syringes.46
The type of glue and method of curing was not spec-
ified in the FDA report but presumably other pro-
cess parameters could impact the leaching of solvents
from needle adhesives and should be considered for
commercial container-closure configurations.
Silicone Oil
Silicone oil is used to lubricate the barrel of glass PFSs
for smooth plunger motion with a lower injection
force. Silicone oil is composed of polydimethylsiloxane
but can also contain trace levels of cyclic polymers
such as octamethylcyclotetrasiloxane (called D4).46
Silicone oil is also used to lubricate stoppers for ease
of handling and smooth insertion into vials.41 In early
studies, adverse effects of silicone oil on protein sta-
bility were reported for the clouding of insulin caused
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
by silicone oil lubricant on syringes.62,63 In a study
of four model proteins, spiking of silicone oil at 0.5%
(v/v) was found to cause increased turbidity in pro-
tein solutions, which was attributed to aggregation.
Interestingly, silicone oil did not cause measurable
perturbation of the structures of the proteins nor alter
their thermal stability.64 Similarly, there were no ma-
jor detectable structural perturbations or decreases
in thermal stability of a mAb spiked with silicone oil
emulsion droplets, presumably because only a minute
fraction of the protein population was adsorbed to the
silicone oil–aqueous solution interface.65 The silicone
oil could adsorb a monolayer of mAb, but formation
of insoluble aggregate particles only occurred when
additional high-speed agitation was applied.65
Formulation conditions resulting in decreased col-
loidal stability of the mAb accelerated aggregation,
and conversely, addition of the surfactant polysorbate
20 completely inhibited aggregation.65 Again, in a dif-
ferent study of four varied proteins (including a com-
mercial mAb and a commercial Fc fusion protein),
spiking of silicone oil into formulations containing
sodium chloride, sucrose, and polysorbate resulted in
an initial protein loss due to surface adsorption with-
out any additional protein loss or aggregation over a
2-week period.66 In this particular study, the silicone
oil effects on protein stability in bulk solution were
minimal (without agitation) even for the range of pro-
tein types and formulation conditions studied.66 The
formulation acted both to alter the adsorption of pro-
tein molecules to the silicone oil droplets and to im-
pact the stability of the silicone oil emulsion itself.66
We note that accelerated stability studies using sili-
cone oil spiking may not be a complete representation
of the actual exposure during transportation and stor-
age of protein in syringes. Agglomeration of silicone
oil droplets could account for some visual observations
of “particles” after exposure to silicone oil: see the il-
lustration for “agglomeration of protein-coated par-
ticles” shown in Figure 1. Polysorbate stabilized the
silicone oil emulsion but also dramatically reduced
adsorption of the protein to the silicone oil droplets,
whereas salt destabilized the emulsion and increased
protein adsorption.66 In a more recent study of two
different mAbs, extensive biophysical and stability
studies showed no change in structure, activity, or
degradation profiles in PFS compared with vials ex-
cept in terms of subvisible particles.67 For the PFS
configuration, there was a small (yet statistically sig-
nificant) increase in the levels of SVPs in PFSs for
both mAbs, most likely due to the presence of sil-
icone oil droplets.67 The authors indicated that the
results of clinical studies for the vial and PFS pre-
sentations indicated no differences in safety, efficacy,
or immunogenicity for patients receiving either the
vial or the PFS presentation.67 Sensitivity to silicone,
DOI 10.1002/jps
 SURFACES AND LEACHABLES EFFECTS ON BIOPHARMACEUTICALS
if it is observed, is likely both protein and formula-
tion specific and may sometimes also require multiple
synergistic factors to occur (e.g., transportation
shocks plus exposure to silicone). The available pub-
lished reports suggest that use of surfactant to min-
imize adsorption to silicone while formulating for
maximum colloidal stability of the protein should
minimize protein–silicone interactions, resulting in
decreased aggregation propensity. Presumably, in-
creased conformational stability should also mini-
mize unfolding at interfaces and therefore also min-
imize potential for aggregation caused by silicone oil
surfaces.
Ongoing developments in syringe siliconization to
optimize and control the level and distribution of sil-
icone oil to maximize functionality while minimizing
the level (and excess) of silicone oil by syringe manu-
facturers may lead to syringes with minimal excess
droplet detachment from the syringe walls during
transportation or over time. Other treatments or sy-
ringe materials or coatings that are being developed
by syringe suppliers could also provide additional fu-
ture configuration options for “silicone oil sensitive”
proteins.
PRODUCT TRANSPORTATION AND STORAGE
Agitation and Air–Water Interface Exposures
Proteins may be exposed to agitation and mechanical
shocks during both production and shipping. Agita-
tion can expose the protein to interfacial forces at the
air–water interface. It has also been reported that
agitation can be a contributing factor to silicone oil
sensitivity.65 Shaking, stirring, or vortexing methods
have been used in accelerated stability testing of pro-
tein formulations. In an agitation study of 13 var-
ied model proteins, particle formation was found to
depend upon both the protein identity and the so-
lution conditions, although no attempts to link the
results with molecular properties were made.68 De-
pending upon the details of the agitation procedure,
vastly different qualitative and quantitative results
may be obtained, most likely because there are mul-
tiple different simultaneous aggregation and particle
formation processes occurring at the air–water inter-
face during agitation. This presents a potential prob-
lem if the formulation was optimized with respect to
a certain accelerated stability test condition but is
exposed to different or additional forces and factors
upon real filling, transportation, and storage. In one
example, stirring of a mAb in Reacti VialsTM (Thermo
Fisher Scientific, Waltham MA) was found to produce
an additional population of smaller aggregates when
compared with horizontal shaking.69 Similarly, stir-
ring was also found to cause more aggregation than
DOI 10.1002/jps
4165
shaking of a different mAb.70 Low-shear exposure to
the air–water interface caused aggregation of recom-
binant human growth hormone but shear acted to en-
hance the rate of aggregation by increasing turnover
of the interface.71 A few studies have reported that
particles, rather than soluble aggregates, were in-
duced by agitation.72–74 These particles slowly dis-
sociated over time and did not nucleate or propagate
further aggregation or particle formation in the bulk
solution, suggesting that they were formed exclu-
sively at the air–water interface.72–74
Although there are numerous examples of where
exposure to the air–water interface caused aggre-
gation and/or particle formation, the fundamental
underlying kinetics and mechanism(s) are still not
completely understood. Aggregation at the air–wa-
ter interface may simply be due to increased num-
bers of collisions between protein molecules at the
interface, leading to the formation of aggregates and/
or particles. The potency of the air–water interface
in inducing aggregation may also be explained in
part by partial unfolding of proteins caused by the
surface tension force at the air–water interface. The
forces at the air–water interface are comparable to
the 20–150 pN forces measured in atomic force mi-
croscopy (AFM) protein unfolding studies, suggesting
that the air–water interface can easily mechanically
unfold sections of proteins (e.g., a 140 pN force is ap-
plied across a 2 nm air–water interface with 70 mN/
m surface tension).19 In the limiting thermodynamic
case, if the surface tension of the interface is greater
than internal forces favoring compaction of the pro-
tein, then the area of the protein must increase (i.e.,
it will unfold) to achieve equilibrium when adsorbed
at the interface.75 The air–water interface is regen-
erative, allowing for cumulative protein damage over
time. Agitation and exposure to the air–water inter-
face involves multiple additional factors. In one study,
it was found that dynamic, dilational, surface area
changes at the air–water interface could cause par-
ticle formation.72 Compression of the interface (re-
duction in the area) by changing the orientation of
vials during gentle rotation showed that compression
of the air–water interface above a critical value of
about five sharply increased particle formation for a
mAb at constant air–water interface area exposure
rate.72 The rate of particle formation was essentially
independent of the bulk protein concentration,72 con-
sistent with a surface-driven process and with other
reports of decreased aggregation on a percentage ba-
sis as bulk protein concentration is increased during
agitation.76
Nonionic surfactants are commonly added to for-
mulations to protect against agitation- or interface-
induced aggregation and this generally appears to
be very effective and rational strategy for many
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
4166 BEE ET AL.
proteins.23,70,77,78 In one review, 13 out of 18 approved
mAb products listed either polysorbate 20 or 80 as
an excipient.79 As described above, accelerated agita-
tion studies are often used to empirically determine
an optimal level of surfactant needed to inhibit ag-
gregation without necessarily elucidating the funda-
mental mechanism(s) of action. Competition for ad-
sorption at interfaces or direct binding to hydrophobic
patches on the protein are two of the most commonly
identified mechanisms for the protective action(s) of
surfactants.75 We note that polysorbate is also an ex-
cipient used to mitigate protein adsorption and un-
folding at other interfaces and may be required at
higher levels to be effective for different surfaces (e.g.,
silicone oil). Agitation studies employing multiple fac-
tors (e.g., air–water interface and silicone oil) are of-
ten used for this purpose. In this section, we focus on
examples relating to air–water interface-induced pro-
tein aggregation and particle formation. For Factor
XIII, maximum protection against agitation-induced
aggregation occurred when surfactant was added at
levels near the critical micelle concentration and
there was no evidence of direct binding of polysorbate
to the protein.28 In contrast, stabilization by direct
binding of polysorbate 20 to hydrophobic patches on
human growth hormone required a ratio of surfactant
to protein of greater than 4 for adequate protection
against agitation, rather than simply requiring lev-
els above the critical micelle concentration (CMC).80
Similarly, polysorbate 20 and 80 were able to protect
AlbutropinTM (Human Genome Sciences, Rockville,
Maryland) against agitation-induced soluble aggre-
gation by binding to the protein at molar ratios of
greater than 9 even when well below the CMC of the
surfactants.81 Polysorbate 20 and 80 could fully pro-
tect darbepoetin alfa from adsorption or agitation-
induced aggregation at about their respective CMC
values.82 Additionally, monomeric polysorbate 20 and
80 also bound to darbepoetin alfa resulting in (differ-
ent) subtle structural changes, yet darbepoetin alfa
did not bind to the polysorbate micelles.82
Some proteins seem quite robust with respect to
agitation: only 0.005% (w/v) polysorbate 20 (again be-
low the CMC) was found to protect a mAb (reported to
be extremely stable with respect to agitation) against
particle formation induced by agitation at both low
and high concentrations even though the “apparent
CMC” in the presence of high concentration mAb was
up to 0.04% (w/v).77 In other cases, proteins are easily
aggregated during agitation, suggesting that suscep-
tibility to aggregation induced by agitation may be
highly protein and formulation specific. In one exam-
ple, the exposure of buried cysteines caused by un-
folding at the air–water interface was found to result
in intermolecular disulfide bonds, possibly resulting
in the formation of a small number of particles that
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 100, NO. 10, OCTOBER 2011
act as nuclei for further particle growth.83 In a similar
way, nuclei for insulin fibrillation were generated by
exposure to the air–water interface during agitation,
followed by further growth in bulk solution.84
Although there are a large number of examples
when surfactants have been extremely effective in
stabilizing proteins against agitation-induced par-
ticle formation, they do not always provide com-
plete protection against all aggregation. For instance,
rather than completely eliminating all aggregation,
addition of polysorbate 80 stabilized small aggregates
of a mAb and prevented their conversion to particles
during agitation studies.69 Surfactants can have “dual
effects on protein stability:”85 Wang et al.85 demon-
strated that polysorbate 80 inhibited the agitation-
induced aggregation of IL-2 mutein, yet it increased
the long-term rate of aggregation and oxidation in
quiescent solutions. In another case, polysorbate ef-
fectively mitigated aggregation for agitated protein
samples, yet again increased the aggregation in qui-
escent samples.76 One reason for this could be in
part because the degradation products of polysor-
bate itself can include species that could potentially
cause protein oxidation.85–87 However, the suscepti-
bility to oxidation and potential for adverse effects
of oxidation is also likely very protein and formula-
tion specific, yet the advantages of polysorbate protec-
tion against agitation-induced particle formation are
great. There may also be other unexpected additional
effects of polysorbate such as enhanced leaching of di-
(2-ethylhexyl)phthalate (DEHP) from polyvinyl chlo-
ride (PVC) intravenous (i.v.) bags discussed in the
Delivery section below. In summary, the literature
shows that polysorbate can be significantly stabiliz-
ing against agitation-induced aggregation of proteins
if added at optimized levels. Control of the quality of
raw materials such as the expiration dates and per-
oxide levels in polysorbate excipients and control of
possible synergistic factors (e.g., Fe ion levels, or oxy-
gen in the headspace of vials, and dissolved oxygen)
could maximize the positive effects of polysorbate as
a stabilizing excipient.
Surfactants are not the only formulation compo-
nent that may adsorb to interfaces or associate with
proteins at interfaces: different buffer ions have been
shown to change the relative affinity of the protein ad-
sorption to the interface and the degree of unfolding
of proteins at the interface.88,89 Cyclodextrin excip-
ients have been reported to have stabilizing effects
on proteins exposed to agitation and air–water in-
terface turnover.90–92 Human serum albumin (HSA)
has been used as an effective excipient for preven-
tion of adsorption losses of low concentration proteins
and also as a stabilizing excipient for very hydropho-
bic proteins.30,93 However, HSA does have multiple
drawbacks as a potential pharmaceutical excipient.94
DOI 10.1002/jps
 SURFACES AND LEACHABLES EFFECTS ON BIOPHARMACEUTICALS
DELIVERY
Syringes, Bags, and Lines for Intravenous Administration
Some proteins may be susceptible to aggregation or
particle formation induced by adsorption to hydropho-
bic surfaces to which therapeutic protein formula-
tions are exposed during delivery of the products to
patients. For instance, exposure to silicone rubber de-
livery tubing decreased IL-2 activity by 97% because
of adsorption to the tubing surface followed by des-
orption of structurally altered inactive molecules.95
Additives to polymers have resulted in adverse in-
teractions with proteins. Plasticizers on the surface
of PVC medical tubing can alter the adsorption of
blood proteins and platelets, leading to thrombus
formation.96,97 Similarly, adsorption of Factor VIII
onto PVC infusion bags reduced its activity by 42%.98
There are different types of i.v. bag materials avail-
able that can be assessed in product compatibility
studies, which are performed by biotechnology compa-
nies to address the International Conference on Har-
monization M4Q guideline, which states that: “The
compatibility of the drug product with reconstitution
diluent(s) or dosage devices (e.g., precipitation of drug
substance in solution, sorption on injection vessels,
stability) should be addressed to provide appropriate
and supportive information for the labeling.”99 Many
products contain specific instructions about handling
and delivery to ensure product quality and stabil-
ity when delivered to the patient. For example, one
brand of antihemophilic factor comes with its own sy-
ringe and tubing for administration and the product
label specifies that the product be delivered within
3 h of reconstitution. The label also notes that the
product “. . .contains polysorbate-80, which is known
to increase the rate of di-(2-ethylhexyl)phthalate
(DEHP) extraction from polyvinyl chloride (PVC).”100
Again, in this case, rather than eliminating the ad-
verse effects on protein stability, addition of polysor-
bate may, counter intuitively, potentially exacerbate
extractions of leachables from PVC containing bags.
In this case, it appears that this potential compatibil-
ity issue was resolved by eliminating the use of PVC.
Of course many of these types of problem are pro-
tein and/or formulation specific and might not be nec-
essary or appropriate for other products. The use of
DEHP has been limited or eliminated in many phar-
maceutical products.41
CONCLUSIONS
Robust control of the manufacturing processes and
unit operations as well as formulation approaches
can often be used to effectively mitigate adverse ef-
fects of interfaces and leachables on protein stability.
For leachables, identification of the molecular species
DOI 10.1002/jps
4167
that has adverse interactions with the protein and
elimination and/or control of its source is one of the
most powerful strategies for improving protein stabil-
ity (e.g., Fe ions and tungsten). Sometimes classifica-
tion of a species as a leachable or an impurity may
complicate identification of the source. Protein insta-
bilities caused by adsorption to equipment surfaces or
due to shed particles can be alleviated by operational
or procedural changes or by changing the surface or
equipment type itself (e.g., membranes, pumps, and
tubing). Similarly, compatibility studies can identify
problems with particular container closures and de-
livery devices. Finally, the behavior of the protein
can be altered using formulation approaches to mini-
mize adsorption losses, aggregation, and particle for-
mation at interfaces (e.g., by reducing propensity for
adsorption).
Exposure to the air–water interface during agita-
tion is a common cause of protein aggregation. As
this interface is ubiquitous, a formulation approach to
minimize its potential effects on stability is often uti-
lized. Addition of nonionic surfactant such as polysor-
bate 20 or 80 appears to be a general and powerful
strategy for protecting proteins against adsorption-
induced aggregation. Polysorbate can also reduce ag-
gregation caused by adsorption to other hydropho-
bic surfaces. Where addition of polysorbate is not
practical or possible (such as before diafiltration), ad-
justment of equipment types and/or operational pro-
cedures and settings could reduce protein particle
formation and aggregation due to large interfacial
area compressions, bubble entrainment, cavitation,
or surface adsorption/desorption enhanced by shear.
Other excipients or solution conditions can also be
used to modulate the interfacial behavior of proteins.
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