REVIEWS

Forced Degradation of Therapeutic Proteins

ANDREA HAWE,1,2 MICHAEL WIGGENHORN,2 MARCO VAN DE WEERT,3 JOERG H. O. GARBE,4
HANNS-CHRISTIAN MAHLER,5 WIM JISKOOT1
1
Division of Drug Delivery Technology, Leiden/Amsterdam Center for Drug Research, 2300 RA Leiden, The Netherlands
2
Coriolis Pharma, Am Klopferspitz 19, 82152 Martinsried-Munich, Germany
3
Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen,
Universitetsparken 2, 2100 Copenhagen, Denmark
4
Analytical Development & Quality Control, Pharma Technical Development Biologics Europe, F. Hoffmann- La Roche Ltd.,
Basel, Switzerland
5
Pharmaceutical & Device Development, Pharma Technical Development Biologics Europe, F. Hoffmann- La Roche Ltd.,
Basel, Switzerland

Received 4 January 2011; revised 17 October 2011; accepted 18 October 2011

Published online 14 November 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.22812

ABSTRACT: The scope of this paper is to review approaches used for forced degradation (syn-
onym, stress testing) of therapeutic proteins. Forced degradation studies play a central role in
the development of therapeutic proteins, for example, for candidate selection, molecule char-
acterization, formulation development, assay development, and comparability studies. Typical
stress methods are addressed within this review, such as exposure to elevated temperatures,
freeze–thawing, mechanical stress, oxidation, light, as well as various materials and devices
used in the clinics during final administration. Stability testing is briefly described as far as
relevant to the discussion of forced degradation studies. Whereas stability-testing requirements
are defined in regulatory guidelines, standard procedures for forced degradation of therapeutic
proteins are largely unavailable, except for photostability. Possible selection criteria to identify
appropriate stress conditions and recommendations for setting up forced degradation studies
for the different phases of development of therapeutic proteins are presented. © 2011 Wiley
Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:895–913, 2012
Keywords: Comparability studies; Forced degradation (stress testing); Analysis; Deamida-
tion; Oxidation; Protein aggregation; Protein formulation; Protein structure; Proteins Stability

INTRODUCTION an acceptable level of impurities, stable and robust

formulations can be reasonably designed to preserve
Therapeutic proteins have acquired a key role in
this state until the final administration to the patient.
the treatment of various diseases such as diabetes,
The robustness of the formulations against external
several types of cancer, and inflammatory diseases.
stress factors is especially important, because thera-
Side effects, immunogenicity, and allergic reactions
peutic proteins are exposed to various types of stress
have been caused when administering rather impure
during production, fill and finish, shipment, stor-
preparations of plasma-derived immunoglobulins,1,2
age, and final administration.6,7 Among these stress
factor VIII,3 and growth hormone from pituitary
factors are temperature changes, elevated tempera-
glands of deceased individuals.4 To prevent such in-
ture, freezing, thawing, mechanical stress, light expo-
cidents, a strong awareness to strive for highly pure
sure, or interaction with specific components derived
and stable protein products has emerged. High purity
from production equipment, primary packaging ma-
begins with production and downstream processing of
terial, or administration devices used, all of which can
therapeutic proteins.5 Only if the bulk material has
potentially impede stability (Table 1).
The scope of our review is to provide an overview
Correspondence to: Wim Jiskoot (Telephone: +31(0)715274314;
Fax: +31(0)715274565; E-mail: w.jiskoot@lacdr.leidenuniv.nl) of forced degradation (stress-testing) methods that
Journal of Pharmaceutical Sciences, Vol. 101, 895–913 (2012) are commonly used during the development of thera-
© 2011 Wiley Periodicals, Inc. and the American Pharmacists Association peutic proteins. Stability testing is briefly addressed,

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012 895

896 HAWE ET AL.

Table 1. Stress Factors a Protein Can Encounter During Real-Life Conditions

Stress Factor Encountered (During Real-Life Conditions)

Elevated temperature Production; improper shipment, storage or handling or deviations; specific production processes (e.g.,
spray-drying)
Freezing, freeze–thawing Accidental freezing during storage or shipment; storage of frozen (bulk) material; lyophilization
Mechanical stress Production (pumping, filtration, stirring, and so on); shipment; handling (e.g., shaking)
Light Exposure to daylight or artificial light during production, shipment, storage or handling; UV detection
during downstream processing
Oxidative stress Contact with oxygen (air, dissolved O2 ); excipients, for example, peroxide impurities in polysorbate;
metal ion traces from production equipment or excipients; light; vaporized sanitation agents, for
example, vaporized hydrogen peroxide; cavitation
pH changes Production (downstream processing); freezing; formulation; dilution in infusion liquids
Interfaces Air–water interface; filters; primary packaging material, for example, vials, stoppers, syringes; infusion
bags and administration lines; silicone oil; extraneous particles
X-ray Air freight transportation

because it is closely related to stress testing at ele-
vated temperatures. Within the review, we use the
following definitions:
r Forced degradation (or stress testing): an um-
brella term covering all forms of applying stress
to drug substance or drug product exceeding the
conditions used for stability testing;
r Stability testing: refers to studies performed to
assess the stability of a formulation according to
the international requirements, in particular In-
ternational Conference on Harmonisation (ICH)
guidelines ICH Q1A8 (for drugs in general) and
ICH Q5C9 (specific for biotech products); and
r Accelerated testing: stability testing at elevated
temperatures under quiescent conditions, ac-
cording to ICH Q1A8 and ICH Q5C.9
Within our review, we discuss the relevance of
forced degradation data for product development, the
importance of suitable analytical methodology, and
factors that could influence the results of these stud-
ies. Test conditions for studying the effect of stress fac-
tors such as temperature, freeze–thawing, mechan-
ical stress, oxidative stress, as well as exposure to
light and various materials on protein stability are
outlined. Because of its importance for successful
product development, we also address compatibility
testing of therapeutic proteins at simulated final ad-
ministration conditions. Finally, we attempt to pro-
vide recommendations and potential selection criteria
for setting up forced degradation studies for therapeu-
tic proteins.
RELEVANCE OF FORCED DEGRADATION
STUDIES FOR THE DEVELOPMENT
OF PROTEIN THERAPEUTICS
Forced degradation studies are an integral part of the
development of therapeutic proteins. Their purpose
may vary depending on the application area and the
phase of development, with a brief overview given in
Table 2.
According to ICH Q5C,9 forced degradation studies
in general can help to (1) assess whether acciden-
tal (or intended) exposure to conditions other than
those proposed, for example, during transportation or
storage, is deleterious to the product and (2) evaluate
test parameters (analytical methods) as indicators of
product stability.
For accelerated testing, ICH Q5C9 states that this
may (1) provide useful data for establishing shelf life,
(2) provide product stability information for future
product development, for example, preliminary as-
sessment of proposed manufacturing changes, such as
changes in formulation or scale-up, (3) assist in setup
and validation of analytical methods for the stabil-
ity program, and (4) generate information that may
help elucidating the degradation profile of the drug
substance or drug product.
During formulation development, forced degrada-
tion studies are essential for the identification of sta-
ble and robust formulations within a reasonably short
time frame. This requires the assumption that the
degradation pathways under the applied stress con-
ditions, for example, at elevated temperature, corre-
late back to the intended storage conditions, which is,
however, not always the case. During formulation de-
velopment, the stability of various formulations dur-
ing forced degradation conditions is compared, more
recently also in high throughput formats.10–12 Using
a combination of stress conditions is of great impor-
tance to identify overall robust formulations, as a
certain excipient might be either stabilizing or desta-
bilizing, depending on the stress factor. One exam-
ple is polysorbate, which can increase the stability of
proteins under agitation conditions, but may lead to
the formation of soluble aggregates and protein ox-
idation under quiescent storage at elevated or even
at the intended storage temperature, especially when
used at low and suboptimal concentrations.13,14 To
ensure protein stability during clinical development

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012 DOI 10.1002/jps
 FORCED DEGRADATION OF THERAPEUTIC PROTEINS 897

Table 2. Relevance of Forced Degradation Studies for the Different Phases of Development, Including Possible Recommendations for
Selecting Suitable Conditions

Application Area Purpose of Study Recommendations

Candidate selection
Molecule characterization
Formulation development
• Select most suitable molecule from a set of
potential candidates
• Elucidate degradation products, pathways,
and mechanisms
• Obtain information about stability of a protein
against various stress factors
• Identify optimum formulation conditions
• Test the robustness of a formulation
• Combine different types of stress factors to gain a
broad insight into several aspects of stability
• Combine different types of stress factors to gain a
broad insight into several aspects of stability
• Include a combination of relevant
(product-dependent) stress factors (e.g.,
temperature, mechanical stress, light stress,
freeze–thawing stress)

Assay development and
validation
Shelf life setting
• Prepare degradation products standards to
develop and validate analytical methods
• Assess stability-indicating properties of
analytical methods
• Establish accelerated degradation profile in
comparison to intended storage
• Predict shelf life
• Choose the stress condition(s) that create(s) the
degradation product of choice for the particular
method
• This will mainly be testing at elevated
temperature; the temperature needs to be
selected based on possible reaction kinetics that
could be applied

Exposure to conditions
other than those proposed
Future product
development/line
extensions
• Support evaluation of accidental (or intended) • Select conditions the protein is expected to be in
exposure, for example, by temperature contact with during production, storage, and
excursion studies, freezing studies, and administration
interaction studies (diluents, container
materials)
• Studies to compare stability behavior of • See formulation development
product, for example, after changes in the
production process (or formulation)
• Collect product stability information

and for commercialization, shipment studies, in-use
studies, and temperature excursion evaluations are
important. Results from accelerated testing can be
used, with certain limitations, for shelf life prediction
of a formulation at 2–8◦ C, as discussed later.
Forced degradation studies go hand in hand with
analytical characterization, because the outcome of
these studies depends on not only the conditions ap-
plied but also the analytical methods included to as-
sess stability. Appropriate analytical techniques that
are capable of detecting and quantifying the different
degradation products formed are essential. As differ-
ent types of degradation products can be formed upon
applying stress to a certain protein15,16 and because
of the complexity of proteins, a combination of comple-
mentary analytical methods is required to cover the
formed degradation products as comprehensively as
possible.17–20 An overview of selected analytical meth-
ods that can be used to characterize different types of
degradation products is given in Table 3 and is fur-
ther discussed in the “Recommendations and Criteria
for Setting Up Forced Degradation Studies” section.
Vice versa, stressing proteins is essential for set-
ting up analytical methods and assessing their sta-
bility—indicating properties (see Table 2). For this
purpose, it is important to create degradation prod-
ucts that are detected by the particular analytical
method(s) of interest. For instance, chemically de-
graded products such as oxidized and deamidated
species may be needed for reversed-phase HPLC
analysis intended for the analysis of these impuri-
ties; for size-exclusion HPLC, the level of aggregates
and/or fragments should be increased. In any case,
these degradation products should be generated in
a controlled manner by exposing the protein to var-
ious stress conditions. Information on the stability
of purified degradation products is crucially impor-
tant if these are routinely used as controls,for ex-
ample, for system suitability testing over a longer
time period. Moreover, the potential effect of sample
preparation, for example, dilution, and the analyti-
cal methods themselves, for example, protein–column
interactions in chromatographic separations, on the
(measured) level of degradation, should be carefully
evaluated.20–22
Upon changes in the manufacturing process of the
marketed product or during development, such as
scale-up, process or cell line changes, the potential
impact of the change on product quality needs to be
assessed. Typically, such comparability exercises in-
clude the analytical characterization of the product
including the analysis of process- and product-related

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
898 HAWE ET AL.
Table 3. Selection of Analytical Methods to Assess Different Types of Degradation Products
Type of Degradation
Soluble aggregates (dimers, trimers, oligomers)
and fragments
Subvisible aggregates, nanometer size range
Subvisible aggregates, micrometer size range
Visible particles
Secondary structure
Tertiary structure
Changes in hydrophobicity
Chemical changes (e.g., oxidation, deamidation)
Abbreviations: AF4, asymmetrical flow field flow fractionation; AUC, analytical ultracentrifugation; CD, circular dichroism; DLS, dynamic light scattering;
HIC, hydrophobic-interaction chromatography; IEF, isoelectric focusing; MALLS, multiangle laser light scattering; NTA, nanoparticle tracking analysis; SLS,
static light scattering; and TDA, Taylor dispersion analysis.
impurities. Furthermore, the stability of the pre- and
postchange product(s) is compared by storage at real-
time and accelerated storage conditions and based on
forced degradation studies. If substantial differences
are found during analysis with a potential impact on
the efficacy or safety of the drug product, further stud-
ies may be necessary such as nonclinical or clinical
studies. The comparability assessment is typically set
up case–by case, depending on the product, its mode
of action, and the type of change made, for exam-
ple, during the manufacturing process. Accelerated
and stress-stability studies (ICH Q1A8 and Q5C9 ) are
often used to establish degradation profiles and pro-
vide a further direct comparison of prechange and
postchange product (ICH Q5E23 ).
WHICH STANDARD CONDITIONS FOR FORCED
DEGRADATION CAN BE FOUND?
In contrast to stability testing, which is well regulated
in the guidelines ICH Q1A8 and ICH Q5C,9 hardly any
standard procedures for forced degradation of thera-
peutic proteins are available. Within the guidelines
a rough concept on forced degradation studies is out-
lined, but information on how and to which extent
these studies should be performed are not provided.24
An exception is photostability testing, where condi-
tions are defined in the ICH guideline Q1B.25 Never-
theless, for biotechnological products, it is proposed
to consider testing conditions others than those de-
scribed in ICH guideline Q1B for photostability test-
ing on a case-by-case basis and to consult the regula-
tory authorities (ICH Q5C).9
With respect to forced degradation, the general
ICH guideline on stability testing (Q1A) recommends
studying the effect of temperature, humidity where
appropriate, that is, for dried preparations, oxida-
tion, and photolysis on the drug. Furthermore, the
susceptibility of the drug to hydrolysis across a wide
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
Selected Analytical Methods
Size-exclusion HPLC, AF4, AUC, electrophoretic techniques (SDS-PAGE,
capillary electrophoresis)
DLS, NTA, AF4, TDA, turbidity/nephelometry, SLS, MALLS
Light obscuration, flow microscopy techniques (e.g., microflow imaging, flow cam),
Coulter counter, light microscopy, fluorescence microscopy, SLS, MALLS,
turbidity/nephelometry
Visual inspection
Far-UV CD spectroscopy, infrared spectroscopy, Raman spectroscopy
Near-UV CD spectroscopy, intrinsic fluorescence spectroscopy, second derivative
UV spectroscopy, nuclear magnetic resonance
HIC, reversed-phase HPLC, extrinsic fluorescent dyes
(HPLC-)mass spectrometry, peptide mapping, reversed-phase HPLC,
ion-exchange HPLC, IEF (gel system or capillary IEF)
pH range should be evaluated.8 ICH Q5C mentions
several stress factors a product may be sensitive to,
which are temperature changes, oxidation, light, ionic
strength, and shear9 ; however, detailed instructions
on how to test the influence of these stress factors on
stability are not available in the guideline.
The American Society for Testing and Materi-
als (ASTM) provides guidance on testing shipping
containers and products in shipping containers in
general.26,27 This information could be used as guid-
ance for mechanical stress testing, as discussed later.
Overall, the lack of predefined standard conditions
for forced degradation studies in the pharmaceutical
field leaves it to each company/laboratory to identify
suitable forced degradation setups for the protein of
interest. Possible approaches range from performing
forced degradation according to standard procedures
defined within the company to setting up stress condi-
tions case–by case for each molecule,24 also depending
on the intended purpose (Table 2).
OVERVIEW OF METHODS FOR FORCED
DEGRADATION STUDIES
Thermal Stress
Proteins can be exposed to temperatures higher than
the recommended storage temperature during pro-
duction, shipment, storage, and final administration.
Elevated temperature is the most widespread method
to stress and degrade therapeutic proteins, which
are typically stored under refrigerated conditions
(2–8◦ C).
With increasing temperature, proteins may un-
dergo conformational changes such as unfolding or
partial unfolding. These changes may subsequently
lead to other degradation reactions, including aggre-
gation. Starting from the onset temperature of un-
folding (Tonset ) toward the melting temperature (Tm )
DOI 10.1002/jps

of the protein, a gradual destabilization of the native
protein structure becomes apparent,28–30 which often
leads to (irreversible) aggregation. At the same time,
diffusion becomes faster at higher temperature, re-
sulting in more energetic collisions with other protein
molecules, as well as with reactive chemicals, thereby
favoring both aggregation and chemical degradation
reactions. Moreover, chemical degradation reactions
may be altered in the unfolded state, for example, if
buried amino acids become accessible for the chemical
reaction in solution.
When setting up the conditions for thermal stress-
ing, the parameters temperature, duration, and op-
tionally temperature fluctuations need to be estab-
lished. The protein’s Tonset and Tm as measures of
the temperature-dependent conformational stability
can be used as rough guidance for selecting an ap-
propriate stress temperature for isothermal incuba-
tion. The Tm is clearly defined as the temperature
where half of the protein molecules are unfolded dur-
ing a thermal scan, assuming a fully reversible un-
folding process,31 whereas the Tonset as the onset
temperature of unfolding is less well defined. Differ-
ential scanning calorimetry,31–33 circular dichroism
(CD),34 FTIR,35 and intrinsic36 and extrinsic37 fluo-
rescence spectroscopy can be used to determine the
Tm and Tonset of a protein. Thermal unfolding can
be reversible like in the case of lysozyme, which un-
dergoes a simple apparent two-state transition un-
der most formulation conditions.38 For the majority
of proteins, thermal unfolding is irreversible, because
it is accompanied or followed by aggregation, as in
the case of interferon-"2a.39 Larger, multidomain pro-
teins exhibit several distinct, in part overlapping un-
folding transitions corresponding to unfolding of the
different domains. For immunoglobulin G (IgG), Fab
and Fc have been shown to denature independently
at 63–71◦ C and 73–82◦ C, respectively, with the tran-
sition as a whole being irreversible.34,40,41
In the context of forced degradation, it should be
noted that unfolded molecules are already present
below the Tm and that the predominant degrada-
tion reaction may depend on the stress temperature.
In particular for aggregation this is the case, as a
number of thermodynamic and dynamic parameters
contribute to the observed aggregation mechanism
and kinetics.42 This is illustrated by the example
of the temperature-dependent aggregation behavior
of IgG.43,44 At temperatures below the first thermal
transition (Tm1 at about 63◦ C), slow aggregation rates
are typically measured,34 as a result of comparably
low levels of partially unfolded IgG molecules with
aggregation mainly being driven by an association of
the Fab domains.45 Incubation at or above the second
thermal transition (Tm2 at about 73◦ C) creates higher
levels of unfolded IgG molecules, leading to more se-
vere aggregation and a different aggregate structure
DOI 10.1002/jps
FORCED DEGRADATION OF THERAPEUTIC PROTEINS 899
involving both Fab and Fc.34 Furthermore, the ag-
gregation pathway and kinetics of thermally stressed
IgGs can vary per IgG type46 and is formulation de-
pendent.
In some cases, the Tm has been used to predict sta-
bility of liquid formulations at the anticipated storage
conditions. Some examples in the literature suggest
a correlation between the Tm and storage stability,
which allowed identifying more stable formulations
for these cases.32,33,47–50 In general, however, Tm mea-
surements cannot replace stability testing under real-
time and real-temperature conditions. For example, if
the predominant degradation process or rate-limited
step is not related with (partial) unfolding of the pro-
tein, the Tm will not be predictive for stability.51,52
Besides the Tm , the reversibility of the unfolding re-
action (i.e., the relative extent of refolding upon low-
ering the temperature below the Tm ) can be used to
predict the stability of some proteins at temperatures
below the Tm , as shown for the recombinant human
Flt3 ligand.53
Numerous attempts have been made to predict the
shelf life of peptide and protein formulations based on
extrapolation of stability data from elevated temper-
ature to real-time storage conditions. This would help
to speed up product development and to avoid waiting
for real-time/real-temperature data. Obviously, this
approach has a major caveat: In all cases, the inves-
tigator needs to consider whether the testing setup
is really predictive for long-term storage under real-
time conditions. This would be the case for degra-
dation reactions following specific reaction kinetics
with predictable increased reaction rates under the
accelerated condition, such as (pseudo) first-order re-
actions following the Arrhenius equation (Eq. 1):
kobs = Ae−Ea /RT (1)
where kobs is the degradation rate constant in day−1 ,
A is the preexponential factor (day−1 ), Ea is the acti-
vation energy in J mol−1 , R is the molar gas constant
(8.314472 J K−1 mol−1 ), and T is the temperature in
kelvin.
In principle, individual chemical degradation reac-
tions can largely be described by the Arrhenius equa-
tion, allowing a calculation of activation energies and
half lives, for example, at the intended storage tem-
perature. However, if several chemical reactions are
occurring in parallel, as is often the case in protein
molecules, the applicability may be limited. Arrhe-
nius would then only be valid to the individual reac-
tions as long as these are not interacting. Examples
of the validity of the Arrhenius equation for chemi-
cal instability are chemical degradation of peptides,
for example, deamidation of oxytocin at low pH,54 hy-
drolysis (deamidation) of insulin,55 and Met oxidation
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
900 HAWE ET AL.
in interleukin-1 receptor antagonist and peptide frac-
tions thereof.56
However, the majority of complex degradation
and aggregation processes in solution exhibit non-
Arrhenius behavior, by definition when the reaction
does not follow (pseudo) first-order kinetics.57–59 Re-
combinant factor VIII SQ (r-VIII SQ) degradation
followed pseudo-first-order kinetics, with nonlinear
Arrhenius plots, which would lead to an overestima-
tion of shelf life at 2–8◦ C based on the data gath-
ered between 10 and 37◦ C, when assuming linearity.57
An example of the successful prediction of the shelf
life for non-Arrhenius-type aggregation is that of re-
combinant bovine granulocyte colony stimulating fac-
tor, where extended Lumry–Eyring models enabled
shelf life prediction from stability data from tem-
peratures between 30 and 47◦ C.58 Another exam-
ple is that of the denaturation of $ galactosidase in
solution59 or aggregation of monoclonal antibodies in
liquid formulations.60
Not only temperature but other factors like pH,
which can also change with temperature, excipients
or protein concentration, may alter degradation rates
and mechanisms.61 Additional factors that may com-
plicate extrapolation approaches include (i) the in-
fluence of surface-induced processes on the aggrega-
tion kinetics, (ii) aggregation processes involving a
lag time prior to a highly variable onset of severe ag-
gregation, (iii) acceleration of aggregation processes
due to the presence of aggregate species function-
ing as nuclei, (iv) temperature-dependent conforma-
tional changes leading to altered reaction kinetics,
and (v) inherent heterogeneity of the pharmaceutical
protein.42 For a detailed overview of principles and ap-
proaches for predicting protein aggregation rates and
shelf life, the interested reader is referred to Weiss
et al.42
A suitable temperature for thermal stress testing
needs to be selected on a case-by-case approach. For
a drug product intended for storage at 2–8◦ C, ac-
celerated testing is generally performed at 25◦ C, as
laid down in ICH Q1A.8 At the same time, the influ-
ence of heat is suggested being tested in increments
of 10◦ C above the accelerated testing temperature,8
which might in some cases be too extreme for sen-
sitive proteins. As a rule of thumb, one should stay
below Tonset and at least 10–20◦ C below the Tm dur-
ing thermal stress testing. At or above the Tm , severe
aggregation often impedes useful conclusions on sta-
bility. Moreover, as the protein will be largely unfolded
under these conditions, degradation products may be
formed that are less relevant to “real-life” conditions.
When setting up provisional shelf life (expiration
dating) during clinical phases, data from accelerated
testing and thermal stress testing can be used in con-
nection with real-time/real-temperature data. How-
ever, the prediction of real-time aggregation rates
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
and shelf life from accelerated data may become pos-
sible only if aggregation kinetics under accelerated/
thermal stress and real-time conditions are qualita-
tively controlled by the same physicochemical factors,
for example, formation of a conformationally changed
intermediate or a chemical change as starting point
for aggregation.42 Therefore, it is important to elu-
cidate degradation mechanisms at thermal stress
conditions and compare them to those occurring at
the intended storage condition, in most cases 2–8◦ C.
The assessment of degradation kinetics can also help
comparing degradation pathways for comparability
exercises.
Incubation in the Frozen State and Freeze–Thawing
Isothermal incubation in the frozen state,
freeze–freeze experiments, and freeze–thawing
experiments are useful to (i) test the stability of
frozen bulk drug substance or early (pre-)clinical
drug products, (ii) assess the robustness of a product
intended for refrigerated storage (2–8◦ C) against
accidental freezing, and (iii) to identify excipients
that stabilize during freezing (also as step in
lyophilization) or frozen storage.
During freezing and thawing, proteins are exposed
to a combination of stress factors,62 including in-
terfacial stresses (protein/ice surface),63–66 tempera-
ture fluctuations,67 cryoconcentration,64 crystalliza-
tion of excipients,68–70 phase separation,71,72 and pH
shifts.65,73–78 The relative importance of each of these
factors with respect to protein instability depends
on the protein, the formulation, and the freeze–thaw
process, and thus needs to be evaluated on a case-
by-case basis. For several proteins, it has been de-
scribed that freeze–thawing may induce the forma-
tion of larger aggregates and proteinaceous particles,
often characterized by a high degree of native-like
protein structure.63,65,66,78–81
Isothermal incubation of a frozen protein formula-
tion at temperatures between–70◦ C, and the thawing
temperature of the product can in some cases provide
suitable stability information and elucidate the influ-
ence of cryoconcentration and excipient crystalliza-
tion on stability. Freeze–freeze studies (temperature
excursions in the frozen state) can simulate the influ-
ence of temperature fluctuations in the frozen state on
the product relevant, for example, when a bulk prod-
uct is stored at–20◦ C and is shipped at lower tem-
peratures. However, the short-term stability of for-
mulations at, for example,–20◦ C will not necessarily
correlate with the stability at lower temperatures
during long-term storage. For instance, the effect
of excipient crystallization and cryoconcentration
is rather temperature specific, rendering stabil-
ity extrapolations to other temperatures a signifi-
cant challenge.68 Nevertheless, incubation studies in
the frozen state at temperatures other than those
DOI 10.1002/jps

intended for storage, as well as freeze–freeze studies
may be useful to test for and/or explain instability as
a result of fluctuating temperatures, which can occur
during shipment or storage of frozen drug substance.
The scale of the experiments is a critical param-
eter, as freezing behavior at large scale is highly
different from small scale.82,83 Small volumes (e.g.,
1–5 mL) can be homogeneously frozen within min-
utes at–20 or–80◦ C. Freezing of larger volumes as
typically applied to storage of drug substance may
require several hours72,82,83 and may result in a
heterogeneous frozen product with respect to pro-
tein and excipient concentration, pH, and osmolar-
ity. To elucidate such cryoconcentration effects, one
can collect samples from various positions in the
container of the frozen formulation, or of the liquid
formulation directly after quiescent thawing, prior
to homogenizing.68,72,75 In most cases, a significant
increase in protein and excipient concentration to-
ward the middle and the bottom of the container has
been found in larger containers.68,72,75 Extrapolation
from small-scale freezing experiments to large scale
is therefore not necessarily possible.
Freezing studies can be carried out to inves-
tigate excipient crystallization at low tempera-
tures. Excipients such as trehalose,68,69 sorbitol,70
or mannitol,84,85 and buffer salts65,73–78 can crystal-
lize in an uncontrolled manner during frozen stor-
age, potentially compromising stability. Sugar crys-
tallization mainly occurs when the formulations are
stored above the glass transition temperature of the
maximally freeze-concentrated solutions (Tg  ). For in-
stance, trehalose was found to crystallize above its
Tg  of–29◦ C during frozen storage, which resulted in
a slow increase in aggregation of a monoclonal anti-
body stored at–20◦ C.68
During freeze–thawing studies, a large number of
process- and formulation-related variables can influ-
ence protein stability. Process-related variables in-
clude the cooling rate, temperature set at start and
end of the freezing process, container geometry, and
filling volume, which will all influence the actual
freezing rate of the formulation. Formulation-related
factors can be among others protein concentration,
pH, and excipients. All of these parameters can have
an effect on the freezing behavior and ice formation,
which is a nucleation-governed process and thus typ-
ically heterogeneous and difficult to reproduce. De-
gree of supercooling and ice crystallization rate are
in general the most important factors controlling the
amount and size of the formed ice crystals and the
dimension of the interfacial area between ice and
freeze–concentrated solution.62
Cooling rates used to freeze the studied formula-
tions can be classified as slow (<1◦ C/min), interme-
diate (∼5◦ C/min), and fast (10–900◦ C/min).62 Note
that the actual freezing rate and the resulting tem-
DOI 10.1002/jps
FORCED DEGRADATION OF THERAPEUTIC PROTEINS 901
perature profile in the container are typically differ-
ent from the externally applied cooling rate used to
freeze the formulation. The resulting freezing rate of
the product depends on various factors, such as heat
transfer to the product, filling volume, container, and
formulation composition,. Fast freezing, for example,
quench cooling in liquid nitrogen or by placing small
volume vials in a freezer at–70◦ C, leads to the for-
mation of many small ice crystals and consequently
a large interfacial area. Depending on the sensitiv-
ity of the studied protein toward surface-induced de-
naturation, this may be detrimental for stability. The
thawing protocol with respect to thawing rate, thaw-
ing temperature, and agitation can also influence the
stability of the formulations. For the model proteins,
lactate dehydrogenase, alcohol dehydrogenase, and
catalase, the highest activity recovery and stability
has been found upon slow freezing (<1◦ C/min) in com-
bination with a fast thawing process (>10◦ C/min),
as the relative surface area was lowest under these
conditions.86
In the context of formulation studies, freeze–thaw-
ing experiments aim to assist selecting the formu-
lations that are stable against accidental freezing
or suitable for lyophilization. Freeze–thawing exper-
iments are often performed by placing the formula-
tions in small containers (1–10 mL, downscale model)
into a freezer (mainly–20 or–80◦ C),87 freezing the
formulations in liquid nitrogen,88 or on the shelf of
a freeze-drier at a controlled cooling rate. Homoge-
neous freezing behavior and limited cryoconcentra-
tion effects are expected for such small volumes, as
discussed above.
An illustrative example of the complex interrela-
tion of various parameters on protein stability dur-
ing freeze–thawing has been published by Kueltzo
et al.64 In this study, the influence of pH, ionic
strength, protein concentration, cooling and thawing
rates, container type and container material on ag-
gregation, and structure of an IgG2 was investigated.
At pH 4, the protein at relatively low concentration
(0.25 mg/mL) was more prone to aggregation dur-
ing freeze–thawing than at a higher concentration
(10 mg/mL), pointing at surface-induced denatura-
tion as a predominant mechanism. The opposite was
found at pH 3, where perturbations of the tertiary
structure in solution and not surface-induced denat-
uration were identified as the cause of aggregation.
In addition, the variation in monomer recovery and
aggregation levels in formulations freeze–thawed in
containers differing in material (glass, polyethylene,
TeflonTM ) and geometry (vials, FlexboyTM bag) il-
lustrate that the container surface can additionally
change the outcome of the freeze–thawing studies.64
How to decide on suitable conditions for
freeze–thawing experiments, incubation at low tem-
perature or freeze–freeze studies? In the simplest
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
902 HAWE ET AL.
case, the placement of vials filled with the liquid
formulation into a freezer might be enough to gain
insight into the general robustness of the protein
against freezing, without specifying freezing or thaw-
ing rates. However, freezing and thawing conditions
are preferably specified and controlled, for example,
by using a cooling/heating bath or a piece of equip-
ment to achieve adequate process control, especially
when applying these conditions routinely during for-
mulation screening for a comparative purpose. Also
in the field of lyophilization process control during
freezing, for example, by using a defined cooling rate
of the shelves in the freeze-drier is important. In both
cases, the resulting freezing and thawing rates within
the formulation could, for example, be measured by
thermocouples placed within the containers. By us-
ing multiple freeze–thawing cycles during formula-
tion screening, the damage caused to the protein can
be exacerbated, enabling a better discrimination be-
tween poor and stable formulations.87,88
The scale of the experiment is crucially important
in all cases, because of the discussed differences in
freezing time, heterogeneity after freezing, as well as
crystallization effects. To study the influence of excip-
ient crystallization on protein stability, both incuba-
tion above and below the Tg  should be carried out. Ex-
cipient crystallization often cannot be predicted easily
as it is considered to be a statistical event more likely
to happen in larger volume.69,70 Therefore, it is sug-
gested to assess nucleation of excipients in the frozen
state, with models that use seeding of freezing nucle-
ation in small scale to induce crystallization.89 Next to
methods for protein stability testing, physicochemical
characterization of the excipients’ morphology, for ex-
ample, by differential scanning calorimetry and X-ray
diffraction, is required.
Mechanical Stress
As therapeutic protein formulations are exposed to
mechanical stress during manufacturing, transport,
and final administration, it is crucial to test their
robustness against these stress factors. Mechanical
stress testing is often performed to evaluate the sta-
bilizing properties of excipients, such as surfactants
added to formulations with the intention of reducing
agitation- and surface-induced aggregation.14,90 Me-
chanical stress testing can be performed as agitation
(shaking)14,90–97 or stirring studies,94,95,98–101 but also
pumping,5,102–104 vortexing,105–107 sonication,108,109
and special shearing devices110–114 have been used for
this purpose.
During mechanical stress testing, proteins expe-
rience several types of stress factors, such as shear-
ing, exposure to liquid–air and liquid–container inter-
faces, cavitation, and local warming effects,18 which
can result in physical and chemical instability. The
relevance of the different stresses on protein stability
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
depends on the experimental setup, the sensitivity of
the protein against mechanical stress, and the formu-
lation.
Proteins are exposed to shear during processing
steps, such as pumping, filtration, or mixing. In most
cases, shear itself was not a major factor with respect
to aggregation induction, even at very high shear
rates of 250,000 s−1 .113,115,116 For typical processing
steps such as pumping or filtration maximum shear
rates of about 20,000 s−1 (shear force of 0.06 pN) have
been estimated.116 To unfold proteins, shear forces
between 20 and 150 pN are required, which would
equal shear rates of up to 5 × 107 s−1 . In compari-
son, upon contact with air–liquid interfaces, proteins
are exposed to forces of about 140 pN, clearly in the
required range for unfolding.116 These estimations
confirm the low aggregation propensity of proteins in
pure shear experiments and point toward other fac-
tors than shear being responsible for unfolding and
aggregation. Adsorption and unfolding of protein at
liquid–air and liquid–solid interfaces, enhanced re-
newal rate of the air–liquid interface, cavitation, air
bubble entrainment, and contamination with parti-
cles or leachables from shearing devices have been
proposed as causative factors for aggregation and in-
stability during mechanical stress testing.111,114,116
The occurrence of minor conformational changes in-
duced by shear stress has been reported only in
few cases, for example, for lysozyme110 and rhGH.113
Overall, in shear stress experiments, the combina-
tion of various stress factors associated with apply-
ing shear appears to be critical. The relative contri-
bution of these factors will strongly depend on the
experimental conditions used in mechanical stress
studies, including parameters such as container
material, container geometry, and interface/volume
ratio.
The extent of the air–liquid interfaces created dur-
ing mechanical stress testing experiments has a huge
impact on the outcome of mechanical stress studies.
Upon contact of a protein with air–liquid interfaces,
(partial) unfolding followed by irreversible aggrega-
tion and/or particle formation can occur.6,7,18 Con-
sequently, one can expect more severe aggregation
during agitation of vials with air–headspace as com-
pared with agitation of fully filled vials.94 Also vari-
ations in the dimension of the liquid–air interface,
for example, by using different filling volumes or vial
sizes, can alter the extent of aggregation or particle
formation.92,94 Furthermore, parameters such as the
velocity of the agitation movements (mainly specified
in rounds per minutes, rpm), the type of movement,
the placement of the vials in the agitation device, tem-
perature, and duration of the agitation stress have an
influence on the outcome of the stress studies. Foam-
ing as a consequence of protein unfolding during me-
chanical stress testing is another factor influencing
DOI 10.1002/jps
 FORCED DEGRADATION OF THERAPEUTIC PROTEINS 903

the properties of the air–liquid interface and was re-

Shaking: 1-mL filling volume, 120 shakes per minute Duration: 70 h

with best stability discrimination: 1-mL protein solution in 2-mL

-coated stirring
Shaking: various filling volumes; 150, 200, and 250 rpm conditions

magnetic vanes Stirring (improved): 30 min at 50 rpm followed

ported to potentially induce oxidation.117

200 rpm at 9-mL filling volume without a headspace Shaking:

Shaking: 0.6-mL filing volume; 350 rpm with vials horizontally
The chosen experimental setup for mechanical

-coated stirring bars at

Stirring (conventional): 30 min at 50 rpm with bottom sitting

Shaking: 4-mL filling volume, 150 rpm horizontally shaking
stress testing depends on the phase of development

2.5-, 5.3-, and 9-mL filling volume at 200 rpm with vials
and the aim of the study. A selection of conditions
and experimental setups of mechanical stress testing

Stirring: 2-mL filling volume, triangular Teflon R

by 15 min at 150 rpm with top-entering stirrer)

found in the literature is provided in Table 4. Stirring
and pumping experiments can be relevant to evaluate
the influence of a manufacturing unit operation, for

Conditions
example, mixing or filling, on protein stability during

horizontally placed Duration: 168 h

Stirring: with 6 mm × 15 mm Teflon R
process development.5,99,100 In that case, the param-

vials at 200 rpm Duration: 120 h

eters should be selected to be a representative of the

bar, 600 rpm Duration: 48 h

manufacturing step of interest.
Agitation (shaking) studies can be performed dur-

placed Duration: 24 h
ing formulation development as a rapid screening test
to discriminate poor from good formulations with re-
spect to their stabilizing power against mechanical
stress. This is in particular relevant during surfac-
tant screening to identify the optimum surfactant
type and concentration. Stirring, which is generally
even harsher than shaking,94,95 may be used for this
purpose as well.
For the simulation of transport-related stress,

6-mL Fiolax vials (shaking)

Examples of Mechanical Stress-Testing Conditions for Therapeutic Proteins Found in the Literature

2-mL polypropylene vials

ASTM provides guidance on testing shipping contain-

and 3-mL Reacti vials

0.8-, 1.5-, 2.0-, and 6-mL
3-mL glass type I vials

6-mL glass type I vials

ers in general.26,27 These include performance test

Stainless steel tanks

Container

glass type I vials

schedules and conditions for testing the integrity of
filled containers in typical shipping environments,
for example, air, rail, or motor transport.26,27 These

(stirring)

(2–10 L)
guidelines are primarily intended for testing the ef-
fect of the applied stresses on the integrity of the con-
tainer itself, and not on protein stability. Nonetheless,
the ASTM guidelines on transport can provide an in- 2–8◦ C and 25◦ C
formative basis on the extent of mechanical exposure
Temperature

a product is subjected to during transport and pos-

sible conditions to simulate this. Suitable equipment
Ambient

Ambient

Ambient

Ambient
to perform studies according to the ASTM guidelines
23◦ C

is available, for example, vibration tables from vari-

ous manufactures,118 which can be applied to testing
the integrity of proteins upon exposure to transport-
Interleukin-2 mutein (5 mg/mL)

(concentration 2–5 mg/mL)

related mechanical stress. Alternatively, mock ship-

Monoclonal IgG1 (10 mg/mL)
Recombinant human growth

Monoclonal IgG (2 mg/mL)

ments can be considered and may include analysis of

Several monoclonal IgGs

product integrity.
hormone (5 mg/mL)
Protein

Exposure to Different Materials

Monoclonal IgG1
(10 mg/mL)

Proteins come into contact with various materials/

surfaces such as glass, rubber, or metal during pro-
duction (pumps, tubings, vessels, chromatography
columns, filters, containers), storage (primary pack-
aging material), and administration to the patient
Stirring and shaking93,94

(infusion bags, infusion systems, syringes). Moreover,

Stirring and shaking95

the outcome of forced degradation studies may largely

depend on the container material used to perform
these studies.
Type of Study

Exposure of protein to materials may cause aggre-

Stirring100
Shaking14

Shaking90

Shaking92
Table 4.

gation, particle formation, and other instability re-

actions such as oxidation, if a protein is sensitive
to a certain type of material or leachables thereof.

DOI 10.1002/jps JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
904 HAWE ET AL.
For example, it has been reported that nonproteina-
ceous particulates, for example, stainless steel parti-
cles from pumps, may cause aggregation in therapeu-
tic protein formulations.102,119 Adsorption of protein
to the steel surface, as well as oxidizing effects of met-
als such as chromium oxide or leaching iron ions from
steel, has been found to potentially trigger aggrega-
tion and particle formation.102,119,120 Moreover, ex-
tractables and leachables from filters have been found
to destabilize IgG2 formulations during agitation and
temperatures stress studies.121 For a detailed review
on the influence of various surfaces and leachables on
aggregation, the reader is referred to the review by
Bee et al.122
In this section, the intentional exposure of the pro-
tein to different materials, for example, in form of
spiking studies, at concentrations and/or surface ar-
eas much higher than expected under real-life con-
ditions, is considered as a stress method. Spiking
studies can be useful to test the potential of mate-
rial surfaces to provoke protein instability. Note that
elucidating the compatibility of a protein with con-
tainer materials is closely related to, but not the
same as, forced degradation studies in the context
of this review. To study container compatibility, pro-
tein adsorption and/or instability caused by different
materials such as glass,123,124 polypropylene,123,124
polystyrene,125 Teflon,97 and stainless steel126 have
been studied.
To clarify the importance of various surface mate-
rials with respect to protein stability, several types
of studies can be envisaged. Spiking of formulations
with the material of choice, for example, glass, metal,
cellulose microparticles,119 silicone oil,127–129 or solu-
ble metal ions (tungsten130 or iron131 ) has been de-
scribed. In most of these spiking studies, the sur-
face area of particles or silicone oil droplets added
to the protein formulations exceeds the surface area
a protein is expected to encounter in real life. For
instance, microparticles with a total surface area of
60–200 cm2 /mL have been added to a monoclonal
antibody in the context of forced degradation stud-
ies, whereas the surface area of glass vials or sy-
ringes is only about 7 cm2 in total.119 Protein stabil-
ity has been tested upon adding silicone oil in con-
centrations between 0.5 and 2.0% (w/v) to protein
formulations.127–129 Although exposure to silicone oil
is relevant as protein comes into contact with silicone
in prefilled syringes and silicone may also leach into
the product over time, the amount typically present
in prefilled syringes is far less (about 300–400 :g for
a 1-mL syringe) than in these studies. Silicone oil it-
self was found to cause little damage to the protein
during quiescent storage, but did accelerate agitation-
induced aggregation.129 However, silicone oil is not
always detrimental to proteins, as was shown for a
fusion protein of albumin and interferon "-2b.132
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
Contact of a protein with other soluble impurities,
for example, tungsten from pins used for the manu-
facturing of certain prefilled syringes,130 iron leaching
from stainless steel surfaces,131 or impurities present
in excipients,133,134 has also been reported to result
in aggregation. By spiking formulations of two thera-
peutic proteins with different levels and types of sol-
uble tungsten species, it was suggested that various
tungsten compounds (WO3 , H2 WO4 , Na2 WO4 , and an
extract from tungsten pins) typically present in pre-
filled syringes can induce the formation of aggregates
and particles at very low pH (e.g., pH 4) and with sen-
sitive proteins, such as the "-helical protein used in
the study.130
Compatibility of proteins with solid surfaces, for
example, of different primary packaging or container
materials, can be tested during accelerated and real-
time stability studies of the actual product. Condi-
tions of testing need to be carefully evaluated in a
case-by-case approach. In a study by Majumdar et al.,
the effect of siliconized and modified syringe surfaces
on the stability of different proteins (among others
a protein with known sensitivity toward silicone oil)
was assessed under quiescent storage at 2–8◦ C and
25◦ C, as well as agitation after storage.135 As another
example, compatibility with stainless steel surfaces
can be evaluated in downscaled stainless steel mini
tanks, in which the protein formulation is stored at
various temperature conditions for specified and rel-
evant time frames. The surface area-to-volume ratio
in downscaled systems is usually different than the
ratio expected in real life, and calculations of these
ratios should be part of setting up such studies. Prior
to this, the compatibility with stainless steel surfaces
in general can also be tested using steel chips.
In summary, spiking studies with different materi-
als can be part of formulation screening to identify the
robustness of formulations toward contact with impu-
rities such as silicone oil or tungsten. The amount and
type of material spiked to the formulation should be
selected case by case. With regard to testing compati-
bility of a protein formulation with silicone oil, it may
also be advisable to test protein stability in the speci-
fied prefilled syringes (including silicone oil at previ-
ously specified ranges) over the shelf life at real time/
real temperature, as well as accelerated temperature
conditions.
Oxidation Stress
Studying oxidation of proteins is very relevant in
formulation development, as oxidation products may
show altered bioactivity and conformation.136 Protein
oxidation can be caused by peroxides present as im-
purities in excipients (e.g., polysorbates), metal ions
from stainless steel surfaces or present as impuri-
ties in excipients, and residues from aseptic agents
(e.g., used in isolators). Oxidation can also be caused
DOI 10.1002/jps

by light, as discussed later. For a detailed descrip-
tion of oxidation mechanisms relevant to therapeu-
tic proteins, the interested reader is referred to the
literature.137–139 Oxidation stress is often used to cre-
ate degradation products, for example, for assay de-
velopment or for elucidating degradation pathways
and kinetics. The general ICH guideline Q1A rec-
ommends assessing the susceptibility of a drug sub-
stance toward oxidation, without further specifying
the conditions.8
Amino acid residues that are particularly sensitive
to oxidation include His, Met, Cys, Tyr, and Trp, but
also other amino acids such as Arg, Pro, and Lys can
be oxidized under certain conditions.6 The relative
distribution of oxidation products is highly dependent
on the oxidizer used. A good example is the work by Ji
et al. on parathyroid hormone 1–34.140 They used sev-
eral conditions reported for the oxidation of Met, His,
and Trp and found that the various procedures could
lead to significantly different products and distribu-
tion of Met, Trp, and His oxidation products even for
this relatively simple protein. Lou et al. showed that
metal-catalyzed oxidation of a IgG2 monoclonal anti-
body resulted in site-specific oxidation of Met (246),
His (304), and His (427) in the Fc portion of the anti-
body, whereas hydrogen peroxide treatment resulted
in oxidation of four solvent-exposed Met.15
Oxidation reactions can be nonsite specific like in
the case of photooxidation and oxidation involving
free radicals or reactive oxygen species.6 Site-specific
oxidation also involves reactive oxygen species, but
mainly occurs when certain metal ions can bind to
specific sites in the protein, for example, His oxida-
tion in the presence of Cu2+ ions.141,142
One common oxidation agent is H2 O2 and within
the European Pharmacopeia an oxidation procedure
by using 0.005% (v/v) H2 O2 for 20 h at 37◦ C is de-
scribed for interferon-".143 Examples on the use of
H2 O2 from the literature are numerous,140,144 includ-
ing Met oxidation induced by 1 mM (ca. 0.003%) H2 O2
for recombinant human interleukin-1 receptor56 or by
0.005% (v/v) H2 O2 for recombinant human interferon
"-2b.145 By applying mild oxidation conditions (H2 O2
at a molar ratio of 80:1 (H2 O2 :protein) for 4 h at
20◦ C) to recombinant human growth hormone, site-
specific oxidation of the relatively accessible Met14
and Met125 residues under preservation of the na-
tive conformation has been achieved.146,147 Not only
the H2 O2 concentration but also the solution pH can
influence the degree of oxidation, because normally
buried Met residues can become accessible due to pH-
induced conformational changes, as shown for human
"-1 antitrypsin.148 The addition of low amounts of
H2 O2 (e.g., 1–100 ppm) and longer incubation peri-
ods of several weeks or months can be performed to
simulate contamination of a formulation with trace
amounts of H2 O2 used as a sanitization agent. De-
DOI 10.1002/jps
FORCED DEGRADATION OF THERAPEUTIC PROTEINS 905
pending on the temperature used during incubation
with H2 O2 , either hydroxyl radicals or superoxide an-
ion radicals may be formed.149
Besides H2 O2 , sometimes in combination with
certain metal ions, t-butyl hydroperoxide (t-BHP)
and 2,2 -azo-bis(2-amidinopropane) dihydrochloride
(AAPH) have been used as oxidation agents. For in-
stance, t-BHP can be an option to oxidize surface-
exposed amino acids.140,150 For metal-catalyzed oxi-
dation combinations of Cu(II), Fe(II), or Fe(III) with
H2 O2 , O2 and/or ascorbic acid have been used.140 A
combination of Cu2+ /ascorbic acid was, for example,
more effective than Fe3+ /ascorbic acid to oxidize Met
residues in human relaxin.151 Metal-catalyzed oxida-
tion is relevant, as metals can be introduced into the
formulation from various sources, for example, during
production, from primary packaging material or from
excipients. AAPH is not directly an oxidizing agent,
but generates alkyl radicals that catalyze the forma-
tion of reactive oxygen species.140
We recommend that different oxidation protocols
are used, both to generate different oxidized species
and to study the susceptibility of the various amino
acids in the protein to oxidation. H2 O2 is an excellent
starting point, generally mainly yielding methionine
sulfoxide. Addition of metal ions such as Fe(III) or
Cu(II) alongside H2 O2 can show whether site-specific
oxidation may occur. The products here are likely
more diverse and may include significant amounts
of oxidized Cys, His, and Trp. If potentially radical-
forming compounds are known to be used somewhere
in the production process or in the formulation itself,
it is advisable to also test these compounds (also in
higher concentrations than in the process of formula-
tion) in combination with H2 O2 . This will then show
whether stability problems may be expected under
normal conditions. To oxidize surface-exposed amino
acids, the use of t-BHP should be considered.
Light Stress
Protein pharmaceuticals are exposed to light in vari-
ous situations. Examples include UV detection during
purification steps and exposure to artificial or sun-
light during production, shipment, storage, and ad-
ministration of the drug to the patient.118,152 Most
marketed therapeutic proteins are recommended to
be “protected from direct (sun)light” and are sold in
adequate secondary packaging to avoid direct and
long-term light exposure.
Light-induced degradation reactions depend on
protein structure and conformation, formulation com-
position, and primary packaging, as well as wave-
length, intensity, and duration of light exposure.
The peptide backbone, Cys, and the aromatic amino
acids Trp, Tyr, and Phe are structural components
that are most sensitive to undergo photodegradation
upon absorption of light. Furthermore, light-induced
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
906 HAWE ET AL.
oxidation of proteins can be caused by the forma-
tion of reactive oxygen species or accelerated by the
presence of metal ions, for example, photooxidation
of His. Photodegradation primarily leads to chemi-
cal changes in single amino acids, but it can also
lead to multiple cross-linking reactions due to di-
tyrosine or disulfide formation and to noncovalent
aggregation.152 An overview of the underlying mech-
anisms of photodegradation of proteins is given by
Kerwin and Remmele.152
The ICH guideline Q1B specifies the experimen-
tal setup for photostability studies, including light
sources and extent of light exposure.25,153 As a light
source, it is possible to use any lamp-producing light
according to emission standard D65 (outdoor day-
light) and ID65 (indoor indirect daylight standard) as
first option, or a combination of a cool white fluores-
cent lamp with a near-UV lamp as a second option.25
The guideline distinguishes between “forced degrada-
tion testing studies,” that is, to estimate the overall
photosensitivity or elucidate degradation pathways,
with conditions not further specified, and “confirma-
tory testing” to predict the stability. For “confirmatory
testing,” the product should be exposed to at least
1.2 million lux hours in the visible range (400–800 nm)
and at least 200 W h/m2 in the UV range (320–400 nm).25
Interestingly, this setup leaves two parameters to be
selected by the investigator: irradiation energy per
unit of time and irradiation time. In most cases, pro-
tein degradation under these very harsh conditions
is significant and thus the “confirmatory testing” is
prone to fail, resulting in severe degradation and ag-
gregation of the product.118 In this case, the product
should be recommended to be protected from direct
light and adequately packaged. In official documents,
no testing conditions are specified on how to deter-
mine adequate levels of light that a product should be
able to withstand during manufacture, storage and
use.
Several therapeutic proteins, for example, recom-
binant human factor VIII,154 recombinant human
interferon-"2a,155 and a human IgG1 monoclonal
antibody,156 were found to be sensitive to light ex-
posure, resulting in discoloration accompanied by
activity loss154,156 and the formation of various chem-
ical and physical degradation products.155,156 Discol-
oration of the solution and loss of activity was mea-
sured for a monoclonal antibody formulation (100 mg/
mL formulated with histidine, sorbitol, and polysor-
bate 80 at pH 5.5) after light exposure according to
the ICH conditions (1.2 million lux hours in the visible
range and 200 W h/m2 in the range 320–400 nm).156
The extent of light exposure may differ per product,
among others depending on the manufacturing pro-
cess steps and the equipment used, making a stan-
dardization of photostability testing difficult. A basis
for defining conditions for photostability testing can
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
be identifying conditions a specific product is exposed
to, for example, during manufacturing, fill and fin-
ish, and the clinical setup. Parameters such as wave-
length of the light sources and exposure time dur-
ing manufacturing and administration can be the ba-
sis for designing photostability studies as addition to
these required by the authorities.118 Proper controls
are of great importance, for example, light-protected
samples to distinguish between the effect of light and
potential warming on protein stability or placebo con-
trols to distinguish between matrix effects and pro-
tein stability.
Compatibility with Final Administration Conditions
For the clinical use of a protein drug, it is important to
assure its compatibility with the final administration
conditions. This includes testing the stability of the
protein after dilution in carrier solutions for infusion
and the compatibility with delivery devices such as
tubing and infusion bags where losses due to adsorp-
tion could occur.
Besides adsorption, leachables from infusion bags,
such as metals, plasticizers, polymerization initiators,
antioxidants, and other additives, potentially impede
protein stability.157–159
The design of these studies depends on the adminis-
tration conditions intended for the protein drug, such
as possible dilution steps in carrier solutions, rate,
and duration of the infusion, materials used for the
infusion, the use of in-line filters, temperature, and
the overall contact time with the different materials.
Many therapeutic proteins are administered as
continuous infusion at room temperature upon dilu-
tion in a carrier solution, usually 0.9% sodium chlo-
ride (saline) and in some cases 5% dextrose. By do-
ing so, the protein is generally exposed to a less
stabilizing environment, because pH, ionic strength,
protein concentration, and temperature differ from
the ideal formulation and storage conditions, and ex-
cipients/stabilizers are typically significantly diluted.
This may lead to degradation during handling, stor-
age prior to administration, or application of the ther-
apeutic protein in the diluent to the patient. For in-
stance, factor VIII exhibited a greater activity loss
in 0.9% NaCl than in 5% dextrose,154 whereas dilu-
tion of trastuzumab is recommended in 0.9% NaCl
and not in dextrose according to the package leaflet.
This illustrates the importance of determining the
compatibility of a therapeutic protein with different
carrier solutions in a case-by-case approach. Other
examples of the compatibility of therapeutic proteins
with diluents are found in the literature, for example,
for insulin160 and epoetin ",161,162 where stability is
typically monitored at 2–8◦ C and ambient tempera-
ture.
Another point to be considered is the loss of protein
or excipients as a result of adsorption to the infusion
DOI 10.1002/jps

bag material, in-line filters or tubings, and instability
caused by surface-induced denaturation.154,163–166 In
the case of interleukin-2, a reduction in the biological
activity by about 90% within a 24-h infusion program
could be attributed to a minor degree to pure sur-
face adsorption, but mainly to irreversible structural
changes in interleukin-2 during a transient associa-
tion with the catheter tubings.164
To set up studies on testing the compatibility of
the protein with the final administration conditions,
a close collaboration with clinicians and clinical phar-
macists is advisable. The planned administration pro-
tocol, including a route of administration, conditions
for infusion or injection, used materials such as tub-
ing, inline filters, and infusion bags, should be the
basis for the study. Once this is defined, the stability
of the therapeutic protein under these conditions can
be systematically studied. To distinguish between the
influence of the carrier solution and the dilution itself
on stability, it is advisable to include suitable controls,
that is, dilution in the formulation buffer. One should
be aware of potential batch-to-batch variations with
respect to leachables from clinical material even if
provided by the same manufacturer, as reported for
standard polyolfin infusion bags.157 A strategy may
be to first use raw materials of construction and to
classify and evaluate suitability and compatibility of
infusion systems in general prior to testing a range of
infusion systems from different manufacturers.
RECOMMENDATIONS AND CRITERIA FOR
SETTING UP FORCED DEGRADATION STUDIES
Guidelines on how to perform forced degradation
studies of proteins in the pharmaceutical field are
widely lacking, leaving it to the pharmaceutical com-
panies or laboratories to setup the conditions for
stress testing. In any case, the chosen setup should
be based on the particular question to be addressed
and the phase of development. One should be very
cautious in extrapolating the outcome of stress stud-
ies to real-life conditions, as the conditions during
forced degradation studies generally differ from what
a protein is expected to encounter during real-life
conditions. Thus, forced degradation studies cannot
replace stability studies under real-time and real-
temperature conditions.
The analytical characterization of the formed
degradation products forms an integral part of forced
degradation studies. Here, it is highly recommended
to use orthogonal analytical methods, capable of de-
tecting and quantifying the degradation products that
may be formed. There are numerous analytical meth-
ods available for the characterization of proteins and
their degradation products (see Table 3). Ideally, all
these methods should be used during forced degra-
dation studies to assure the most complete charac-
DOI 10.1002/jps
FORCED DEGRADATION OF THERAPEUTIC PROTEINS 907
terization of the degradation taking place. In prac-
tice, this is not possible, as a result of constraints on
time, finances, and available methodology. The scien-
tist must therefore initially select a set of those meth-
ods to explore whether any significant degradation
takes place. Ideally, at least one method for each type
of anticipated class of degradation product should be
used. Depending on the type of degradation products
identified in the first screening, a more in-depth anal-
ysis can be performed. Note that the selection of meth-
ods depends on the forced degradation method, the
protein, and its concentration, and the formulation.
For more information about analytical methods, the
reader is referred to the literature.17–20,167
The chosen formulation conditions with respect to
pH and ionic strength most likely influence the out-
come of forced degradation studies. For certain ques-
tions, the exposure to extreme formulation conditions,
for example, with respect to pH, ionic strength, or ex-
cipient composition/concentration, can be considered
as a stress method to address the robustness of a pro-
tein during processing and formulation. Formulation
pH, ionic strength,6 and excipients133,134,168,169 can
have an influence on chemical stability, as well as col-
loidal stability of a protein.7,18 As an example, the
exposure of a protein to extreme (acidic or basic) pH
values could be used to test a protein’s susceptibility
to deamidation.
Primary packaging material and the scale of the
experiments (meaning the container volume and the
filling volume) may influence protein stability during
forced degradation studies. This can be due to ad-
sorption of the protein to the container, other surface-
induced effects, compounds leaching from the con-
tainer into the formulation, or the influence of fill-
ing volume on the outcome of stress tests (e.g., as
discussed in Freeze–Thawing and Mechanical Stress
Testing sections). Therefore, it is suggested to per-
form forced degradation studies in the final primary
packaging material or the container of interest when
testing for production-related stress. Depending on
the stage of development, this is not always feasible,
in particular in early phases of development when
only limited amount of material is available or when
the protein concentration, volume, and final container
have not yet been defined. In the last example, the
results from forced degradation studies performed
in different containers can help identifying suitable
primary packaging materials for the formulation of
interest.
In some cases, it is less critical to downscale forced
degradation studies and use sample volumes smaller
than the final drug product formulations. When the
aim is creating degradation product standards, the
scale of the experiment and the primary packag-
ing may still affect the characteristics of the degra-
dation products obtained, which is, however, less
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
908 HAWE ET AL.
critical regarding the relevance for the intended us-
age. Also for certain formulation studies, when com-
paring the stability of a protein under different pH
or ionic strength conditions in quiescent storage, full-
scale experiments may not necessarily be required.
Bridging experiments, comparing the stability of a
protein in different primary packaging materials, or
different scales can give insight into the relevance of
these parameters on the outcome of forced degrada-
tion studies.
Selection criteria and recommendations for forced
degradation studies depend on the aim of the study,
with possible criteria summarized in Table 2. In some
cases, for example, when testing the robustness of
formulations, it is advisable to select conditions that
are relevant to real-life stresses if a protein is poten-
tially exposed during production, shipment, storage,
and final administration. To cover as many potential
degradation mechanisms as possible, a combination of
various types of stress conditions should be tested in
separate experiments, for example, temperature, in-
terfaces (ice–water, liquid–air, liquid–container) and
light-induced stress protocols.
To gain insight into product characteristics and/or
create degradation products for assay development,
conditions that are less relevant to real life can still
provide valuable information. Examples can be the
exposure to extreme (both low and high) pH values,
or oxidation by adding chemicals such as H2 O2 . When
creating degradation products for assay development,
it can be useful to select conditions where a certain
type of degradation product is primarily created, for
example, oxidized species by H2 O2 or metal-catalyzed
oxidation. Ideally, it should be possible to create differ-
ent levels of the degradation products in a controlled
and reproducible way. This may also help assessing
a potential further relevance of these impurities. As
an example, by selective oxidation of specific Met
residues—in the absence of other degradation pro-
cesses—one could create specific degradation prod-
ucts and measure bioactivity of these species. When
degradation products cannot be “selectively gener-
ated,” it can be considered to purify them for further
testing. As an example, the isomerization products
of Asp102 and the deamidation products of Asn30 of
trastuzumab were generated by thermal stress and
purified by ion exchange chromatography for bioac-
tivity testing.170 Assessing the stability of purified
degradation products is important, if those will be
routinely used as standards/control solutions for an-
alytical methods or stored for a longer time prior to
activity testing.
During formulation development, the selected
stress conditions should most importantly allow dis-
crimination of stable and unstable protein formu-
lations. For this purpose, sufficiently high levels of
degradation products need to be created within a
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 101, NO. 3, MARCH 2012
reasonable time frame. However, too high levels of
degradation might again impede stability discrimi-
nation. What is sufficiently high in a reasonable time
frame? In the context of forced degradation studies of
proteins, this should be evaluated in a case-by-case
approach. The suggestion to create not more than
5–15% degradation of the main compound in a reason-
able time frame (e.g., at most 2 weeks) as described in
the context of forced degradation studies for small
molecules24 could be a rough guidance for protein
therapeutics. However, because of the complexity of
degradation products formed in protein formulations
this might not be applicable to all types of instability.
Subvisible and visible particles formed upon forced
degradation studies typically represent only a minor
fraction of the overall protein present in the formu-
lation, but are still an important and well-detectable
class of degradation products. The time frame should
make sense for the selected stress conditions and can
range from minutes, for example, for vortexing or
shearing studies, to several days or weeks for shaking
studies or exposure to elevated temperature.
The conditions selected should cover potential
stress conditions a protein is expected to be exposed to
during production, storage, transportation, and han-
dling. Those can vary for different products, depend-
ing on the formulation (e.g., liquid or dried), primary
packaging (e.g., a prefilled syringe or a vial with head
space), and administration conditions (e.g., infusion
after dilution in a carrier solution or direct injection).
CONCLUSIONS
Forced degradation studies play a central role in
the development of therapeutic proteins. In contrast
with the available guidelines for stability studies,
specific guidance for forced degradation studies is
lacking. Stress conditions typically used, among oth-
ers elevated temperature, freeze–thawing, mechan-
ical stress, or light, can induce different types of
chemical and physical degradation reactions in ther-
apeutic proteins. The prediction of real-time/real-
temperature stability from accelerated testing data
is with limitations possible, but should be performed
with care. In general, forced degradation studies do
not replace stability testing. Moreover, they are ap-
plied to many other purposes than stability testing,
such as candidate selection, molecule characteriza-
tion or assay development, and validation.
The analytical characterization of the induced
degradation products forms an integral part of stress
studies. It is of great importance to have suitable an-
alytical techniques available to detect and quantify
degradation products as comprehensively as possible.
By doing so, a thorough understanding of underlying
mechanisms of protein degradation can be generated,
which provides a basis for finding means of preventing
DOI 10.1002/jps

those. Overall, forced degradation studies can provide
important insights into the sensitivity of a protein to-
ward various stress conditions important for the de-
velopment of stable products and user instructions in
package inserts.
ACKNOWLEDGMENTS
This work was supported, in part, by the Dutch
Technology Foundation (Stichting Technische Weten-
schappen, STW), Applied Science Division of Neder-
landse Organisatie voor Wetenschappelijk Onderzoek
(NWO), and the Technology Program of the Ministry
of Economic Affairs. The authors thank Connie van
Gent for the literature supply and the anonymous re-
viewers for their thoughtful, constructive comments.
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