Data and Review
Toward a Generic Approach for
Stress Testing of Drug
Substances and Drug Products
Silke Klick,* Pim G. Muijselaar, Joop Waterval,* Thomas Eichinger, Christian Korn, Thijs K. Gerding, Alexander J.
Debets, Cari Snger-van de Griend, Cas van den Beld, Govert W. Somsen, and Gerhardus J. De Jong
The Impurity Profiling Group has developed a
generic approach for conducting stress testing
on drug substances and drug products.The
proposed strategy is evaluated and verified
with historical data and new experiments.
Results demonstrate that the proposed
approach is reasonable and generates
relevant, generally predictive results for the
development of a stability-indicating method.
S. Klick, PhD, is a team manager, Analytical
Development, at AstraZeneca R&D (Mlndal
Sweden), tel. 146 31 7761758, fax 146 31
7767337, Silke.Klick@astrazeneca.com. P.G.
Muijselaar, PhD, is a senior analytical scientist
and T.K. Gerding, PhD, is a manager, Sector
Chemical & Pharmaceutical Development, at Solvay
Pharmaceuticals (The Netherlands). J. Waterval,
PhD, is a group leader, Department of Pharma-
ceutics at Akzo Nobel, N.V. Organon (The
Netherlands), tel. 131 412662070, fax 131
412662524, Joop.Waterval@organon.com.
T. Eichinger, PhD, is a deputy global director,
Analytical Sciences Department and C. Korn,
PhD, is a manager, Analytical Development,
Sanofi-Aventis Frankfurt (Germany). A.J. Debets,
PhD, is a general manager of Organon
Development GmbH, Akzo Nobel (Germany). C.E.
Snger-van de Griend, PhD, is an associate
principal scientist, Analytical Development, at
AstraZeneca (Sdertlje, Sweden). C. van den
Beld, PhD, is the section head, Pharmaceutical
Development Section/PDD at Yamanouchi Europe
BV (The Netherlands). G.W. Somsen, PhD, is
an associate professor and G.J. De Jong, PhD,
is a professor, Pharmaceutical Analysis, Utrecht
University (The Netherlands).
*To whom all correspondence should be addressed.
48 Pharmaceutical Technology FEBRUARY 2005
S
tress testing is defined as the stability testing of drug sub-
stances and drug products under conditions exceeding
those used for accelerated testing. Although it is an inte-
gral part of the information provided to regulatory au-
thorities in registration application dossiers (ICH Q1A[R2])
(1), details regarding the design of such studies are not covered
by regulatory guidance documents (see Reynolds et al. for a re-
view of regulatory guidance on stress testing [2]).
Pharmaceutical companies perform stress testing (also called
forced-degradation studies) during preformulation to help se-
lect compounds and excipients for further development, to fa-
cilitate salt selection or formulation optimization, and to pro-
duce samples for developing stability-indicating analytical
methods. Stress testing provides information about degrada-
tion mechanisms and potential degradation products. This in-
formation then can be used to develop manufacturing processes
or to select proper packaging. It may also help in preparing ref-
erence material of identified degradation products. Although
preformulation work is part of early-phase drug development,
stress testing often is repeated when manufacturing processes,
product composition, and analytical procedures are refined and
reach a more final state (3). This is usually the case when a com-
pound enters clinical Phase 3 studies. According to various reg-
ulatory guidance documents, results from stress testing should
be included in the dossier when a registration application is
submitted to regulatory authorities.
This article discusses stress testing according to the regula-
tory guidance documents, with emphasis on what should be
considered for late-clinical phases and for registration applica-
tion dossiers (e.g., marketing authorization applications or new
drug applications). (During early development, other aspects
of stress testing may be relevant for various screening purposes,
but these are not the primary subject of this article.)
Many pharmaceutical companies have policies such as stan-
dard operating procedures or internal guidelines for stress test-
ing. There is no consensus, however, about how much stress is
adequate. This fact was recently confirmed in an extensive bench-
marking study in which information about the design, condi-
tions, procedures, and the organization to oversee stress test-
ing activities was requested from 20 companies (3). The authors
concluded that the pharmaceutical companies were applying
significantly diversified approaches.
www.phar mtech.com
Data and Review
Almost no literature can be found in
Overview of stress-testing guidance documents which stress conditions were investigated
Standard Title and reference Status Date on their ability to predict the degrada-
ICH Q1A(R2) Stability Testing of New Drug Substances and Products (1) Step 4 February 2003 tion products formed during long-term
ICH Q1B Photostability Testing of New Drug Substances and Products (12) Step 4 November 1996 stability studies of marketed formula-
ICH Q2B Validation of Analytical Procedures:Methodology (14) Step 4 November 1996 tions. Some direction regarding the prac-
ICH Q3A(R) Impurities in New Drug Substances (15) Step 4 February 2002 tical conduct and issues related to stress
ICH Q3B(R) Impurities in New Drug Products (16) Step 4 February 2003 testing under various ICH prescribed
FDA Guidance Submitting Documentation for the Stability of Human Drugs February 1987 conditions has been published (5). This
and Biologics (6) reference described a classification scheme
FDA Guideline Submitting Samples and Analytical Data for Methods Validation (7) February 1987 and included decision trees to help select
FDA Reviewer Validation of Chromatographic Methods (8) November 1994 the right type of stress conditions. Al-
Guidance though this approach is systematic, the
FDA Guidance Stability testing of Drug Substances and Drug Products (9) Draft June 1998 suggested stress conditions seem rather
FDA Guidance Analytical Procedures and Methods Validation (11) Draft August 2000 harsh. For example, refluxing the drug in
FDA Guidance INDs for Phase 2 and Phase 3 Studies,Chemistry,Manufacturing, May 2003 0.1 N HCl /0.1 N NaOH for 8 h was sug-
and Controls Information (10) gested as a starting condition, which in
ICH Q1A(R2): Stability Testing of New Drug Substances and Products our opinion would generate irrelevant
Drug substance. Stress testing a drug substance can help identify the likely degradation products,which can in degradation in many cases.
turn help establish the degradation pathways and the intrinsic stability of the molecule and validate the Representatives from several pharma-
stability-indicating power of the analytical procedures used.The nature of the stress testing will depend on the ceutical companies and Utrecht Univer-
individual drug substance and the type of drug product. sity (The Netherlands) have collaborated
Stress testing is likely to be carried out on a single batch of the drug substance.It should include the effect of to form the Impurity Profiling Group
temperatures (in 10 8C increments [e.g., 50 8C,60 8C] above that for accelerated testing),humidity (e.g., 75% RH (IPG). For approximately three years, this
or greater) where appropriate,oxidation,and photolysis on the drug substance.Testing should evaluate the group has examined the issues relevant to
susceptibility of the drug substance to hydrolysis across a wide range of pH values when in solution or stress testing of low molecular weight com-
suspension.Photostability testing should be an integral part of stress testing. pounds. The main focus of the group has
Examining degradation products under stress conditions is useful for establishing degradation pathways and been to generate representative degrada-
developing and validating suitable analytical procedures.It may not be necessary,however,to examine for tion samples for developing stability-in-
specific degradation products if previous studies have demonstrated that these products are not formed under dicating analytical methods for both drug
accelerated or long-term storage conditions. substances and drug products. The pre-
Drug product. The design of formal stability studies for a drug product should be based on the behavior and dictive value of the degraded samples for
properties of the drug substance,the results from stability studies on the drug substance,and the experience real-time storage of the final product
gained from clinical formulation studies.The likely changes to storage conditions and the rationale for the under long-term and accelerated storage
selection of attributes to be tested in the formal stability studies should be stated.Photostability testing should conditions was defined as a measure of the
be conducted on at least one primary batch of the drug product if appropriate.Standard conditions for balance between purposeful degradation
photostability testing are described in ICH Q1B. and the formation of irrelevant artifacts
Any evaluation should take into account not only the assay but also the degradation products and other that may lead to misinterpretations (e.g.,
appropriate attributes.Where appropriate,attention should be paid to reviewing the adequacy of the mass secondary degradation products).
balance and stability and degradation performance. This article reviews the regulatory
sidebar continued on page 52 guidances exact text related to stress test-
ing and the current practices in partici-
pating companies and presents a proposal
Many researchers have developed stability-indicating meth- and verification with practical results of the feasibility of a
ods that involve applying various stress regimes to generate de- generic approach toward stress testing.
graded samples. Bakshi and Singh evaluated these studies and
concluded that the majority would fall short of meeting ICH Overview of regulatory guidance documents
and FDA requirements (4). The main issue was that the required Several regulatory guidance documents mention forced degrada-
stress studies were not performed. tion or stress testing of drug substances and/or drug products (1,
Although the development of a broad range of pharmaceu- 616). A summary of these has been published (2). To enable a
tical compounds requires flexibility in how stress testing is good interpretation of the available guidance documents and to
conducted, many companies apply extreme stress conditions. provide the reader with a comprehensive overview, the text of the
Typically, stress conditions are determined on the basis of the relevant paragraphs of these guidance documents is provided in
senior scientists experience or are copied from previously ap- the sidebar, Overview of stress-testing guidance documents.
plied example studies on other products. Rationales explain- The ICH stability guideline Q1A(R2) defines stress testing
ing why certain stress conditions are applied typically are not for drug substances and drug products. For drug substances,
provided. these tests are studies undertaken to elucidate the intrinsic stabil-
50 Pharmaceutical Technology FEBRUARY 2005 www.phar mtech.com
Data and Review
ity of the drug substance. Such testing is part of the
Overview of stress-testing guidance documents (continued) development strategy and is normally carried out
ICH Q1B: Stability Testing: Photostability Testing of New Drug Substances and Products under more severe conditions than those used for ac-
Drug substance. Photostability testing should consist of two parts:forced-degradation testing and celerated testing. For drug products, stress tests are
confirmatory testing.The purpose of forced-degradation testing is to evaluate the overall studies undertaken to assess the effect of severe con-
photosensitivity of the material for method-development purposes and/or degradation pathway ditions on the drug product. Such studies include pho-
elucidation.This testing may involve the drug substance alone and/or the substance in simple tostability testing (see ICH Q1B) and specific testing
solutions and suspensions to validate the analytical procedures.In these studies,the samples should on certain products (e.g., metered-dose inhalers,
be in chemically inert and transparent containers.For forced-degradation studies,various exposure creams, emulsions, and refrigerated aqueous liquid
conditions may be used,depending on the photosensitivity of the drug substance and the intensity of products).
the light sources.For development and validation purposes,it is appropriate to limit exposure and end In addition to investigating drug substance or drug
the studies if extensive decomposition occurs.For photostable materials,studies may be terminated product stability, stress testing studies may also pro-
after an appropriate exposure level has been used.The design of these experiments is left to the vide information about degradation pathways and
applicants discretion although the exposure levels used should be justified. the selectivity of the applied analytical methods. Ac-
Under forcing conditions,decomposition products may be observed that are unlikely to be formed cording to the ICH and FDA guidance documents,
under the conditions used for confirmatory studies.This information may be useful in developing and stress testing is conducted to fulfill three main pur-
validating suitable analytical methods.If in practice it has been demonstrated that they are not poses: to provide a stability assessment of the drug
formed in the confirmatory studies,these degradation products need not be further examined. substance or the drug product; to elucidate the pos-
ICH Q2B: Validation of Analytical Procedures: Methodology sible degradation pathways of the drug substance or
Drug substance/drug product. If impurity or degradation product standards are unavailable, the active pharmaceutical ingredient in the drug prod-
specificity may be demonstrated by comparing the test results of samples containing impurities or uct; and to investigate the stability-indicating power
degradation products with a second well-characterized procedure;e.g., pharmacopeial method or of the analytical procedures applied for the drug sub-
other validated analytical procedure (independent procedure).As appropriate,this should include stance and the drug product.
samples stored under relevant stress conditions (light,heat,humidity,acidbase hydrolysis, Although the regulatory guidance documents
oxidation). listed in the sidebars define the concept of stress
testing, they do not provide detailed information
ICH Q3A(R): Impurities in New Drug Substances
about a stress testing strategy. The experimental
Drug substance. The applicant should summarize the actual and potential impurities most likely to
conditions to conduct stress tests are described in a
arise during the synthesis,purification,and storage of the new drug substance.This summary should
general way and the exact stress conditions to be ap-
be based on sound scientific appraisal of the chemical reactions involved in the synthesis,impurities
plied are not described. Some guidelines emphasize
associated with raw materials that could contribute to the impurity profile of the new drug
that the experimental conditions of stress tests de-
substance,and possible degradation products.This discussion can be limited to those impurities that
pend on the nature of the drug substance and the
might reasonably be expected based on knowledge of the chemical reactions and conditions
drug product (1, 9).
involved.In addition,the applicant should summarize the laboratory studies conducted to detect
The available guidance documents also do not state
impurities in the new drug substance.This summary should include test results of batches
the extent to which stress tests should be carried out
manufactured during the development process and batches from the proposed commercial process,
that is, how much stress should be applied or how
as well as the results of stress testing (see ICH Guideline Q1A on Stability) used to identify potential
much degradation should be aimed for. It is recog-
impurities arising during storage.The impurity profile of the drug substance batches intended for
nized, however, that during stress testing, degrada-
marketing should be compared with those used in development and any differences discussed.
tion products can be observed that are not formed
ICH Q3B(R): Impurities in New Drug Products during accelerated or long-term stability studies.
Drug product. Analytical procedures should be validated to demonstrate specificity for the specified Hence, these degradation products need not always
and unspecified degradation products.As appropriate,this validation should include samples stored to be examined (1, 9).
under relevant stress conditions:light,heat,humidity,acid/base hydrolysis,and oxidation. It can be concluded that the available guidance doc-
FDA Guidance for Industry: Submitting Documentation for the Stability of Human Drugs uments allow for performing stress tests on a sound
and Biologics scientific basis. The experimental conditions should
Drug substance. A program for the stability assessment might include storage at ambient be realistic and lead to purposeful degradation. That
temperature and under stress conditions.Stress testing conditions ordinarily include temperature is, stress tests should generate representative samples
(e.g.,5,50,and 758C),humidity,where appropriate (e.g.,75% or greater) and exposure to various to assess drug substance and drug product stability,
wavelengths of electromagnetic radiation (e.g., 190780 nm UV and visible ranges),preferably in provide information about possible degradation path-
open containers,where applicable.It is also suggested that the following conditions be evaluated in ways, and demonstrate the stability-indicating power
stability studies on solutions or suspensions of the bulk-drug substance:acidic and alkaline pH,high- of the analytical procedures applied.
oxygen atmosphere,and the presence of added substances under consideration for product
formulation. Current practice in participating companies
Drug product. Stress testing the drug product helps identify potential problems during storage and As a starting point for the IPGs discussion about
transportation and provides an estimate of the expiration-dating period. stress testing, participants shared existing strategies
sidebar concluded on page 54 for stress testing applied by the participating com-
panies. The approaches were very different. Some
52 Pharmaceutical Technology FEBRUARY 2005 www.phar mtech.com
Data and Review
Adequate
13%
Out of
Overview of stress-testing guidance documents (concluded) proportion False
FDA Guideline for Submitting Samples and Analytical Data for Methods Validation 10% 34%
Drug product. Information supporting the suitability of the methodology for the dosage form.
Overdone
Generally,this should include the following:A degradation schematic for the active ingredient in the
14%
dosage form,where possible (e.g., products of acidbase hydrolysis,temperature degradation,
Soft
photolysis,and oxidation). 29%
FDA Reviewer Guidance: Validation of Chromatographic Methods
Drug substance. Submission of data from stress testing the drug substance using acid and base
hydrolysis,temperature,photolysis and oxidation according to the Guideline for Submitting Samples Figure 1: The effect of a fixed set of fast and severe
and Analytical Data for Methods Validation is recommended. stress conditions on relevant degradation.
FDA Guidance for Industry: Stability Testing of Drug Substances and Drug Products
companies had standard operating procedures in
Drug substance/drug product. Stress testing helps determine the intrinsic stability characteristics of
place in which conditions for stress testing stud-
a molecule by establishing degradation pathways to identify the likely degradation products and to
ies were described in detail. Other companies were
validate the stability-indicating power of the analytical procedures used.Stress testing provides data
applying case-by-case approaches that depended
about the forced-decomposition products and decomposition mechanisms of the drug substance.The
on the chemistry of the compound to be evalu-
severe conditions that may be encountered during distribution can be covered by stress testing
ated or the dosage form to be developed.
definitive batches of the drug substance.These studies should establish the inherent stability
After thoroughly discussing the various pro-
characteristics of the molecule such as the degradation pathways and lead to identification of
cedures, members of the IPG agreed that the com-
degradation products and hence support the suitability of the proposed analytical procedures.The
panies were applying conditions that in many
detailed nature of the studies will depend on the particular drug substance and type of drug product.
cases were too drastic and were causing irrele-
This testing is likely to be carried out on a single batch of a drug substance.Testing should include
vant degradation. Typical conditions for stress
the effects of temperatures in 10 8C increments above the accelerated temperature test condition
testing in solution included various pH (112),
(e.g., 50 8C,60 8C) and humidity,where appropriate (e.g., 75% or greater).In addition,oxidation and
high temperatures (.70 8C), extreme light expo-
photolysis on the drug substance plus its susceptibility to hydrolysis across a wide range of pH values
sure, or the presence of oxidative or reductive
when in solution or suspension should be evaluated.Results from these studies will form an integral
agents. For stress testing in the solid state, applied
part of the information provided to regulatory authorities.Light testing should be an integral part of
conditions included high temperatures (.70 8C)
stress testing.
and relative humidity (RH) (.70%). These con-
Some degradation pathways can be complex and under forced conditions,decomposition products
ditions frequently are set on the basis of tradi-
may be observed that are unlikely to be formed under accelerated or long-term testing.This
tion rather than scientific rationale.
information may be useful in developing and validating suitable analytical methods,but it may not
To investigate the effectiveness and possible
always be necessary to examine specifically for all degradation products if,in practice,it has been
shortcomings of typical fast and severe stress con-
demonstrated that these are not formed.
ditions, a screening study was set up by one of the
FDA Guidance for Industry: Analytical Procedures and Methods Validation participating companies. Fifteen low molecular
Drug substance/drug product. Degradation information obtained from stress studies (e.g., products weight (i.e., nonbiologically derived) compounds
of acid and base hydrolysis,thermal degradation,photolysis,oxidation) for the drug substance and were studied using a fixed set of 11 commonly ap-
for the active ingredient in the drug product should be provided to demonstrate the specificity of the plied stress conditions (see Table I). To save time,
assay and analytical procedures for impurities.The stress studies should demonstrate that impurities fast (drastic) conditions were applied (e.g., a max-
and degradants from the active ingredient and drug product excipients do not interfere with the imum two weeks at high temperature for solid
quantitation of the active ingredient. stressing and a maximum one day for stress in so-
FDA Guidance for Industry: INDs for Phase 2 and Phase 3 Studies, Chemistry lution). Because no long-term stability data were
Manufacturing, and Controls Information available for the test compounds, accelerated stor-
Drug substance. Performance of stability-stress studies with the drug substance early in drug age conditions (six months at 40 8C and 75% RH)
development is encouraged because these studies provide information crucial to selecting stability- provided the relevant degradation products.
indicating analytical procedures for real-time studies.If not performed earlier,stress studies should The screening study did not include stable
be conducted during Phase 3 to demonstrate the inherent stability of the drug substance,potential compounds in which no degradation products
degradation pathways,and the capability and suitability of proposed analytical procedures.Stress formed under accelerated conditions. The ob-
studies should assess the stability of the drug substance in different pH solutions,in the presence of served degradation was classified into five cate-
oxygen and light,and at elevated levels of temperatures and humidity.These one-time stress studies gories (see Table II).
on a single batch are not considered part of the formal stability program. The category adequate is considered optimal
Drug product. For certain drug products,one-time stress testing can be warranted to assess the and out of proportion is considered acceptable.
potential for changes in the physical (e.g., phase separation,precipitation,aggregation,changes in All the other categories are of no additional value
particular-size distribution) and/or chemical (e.g., degradation and/or interaction of components) and may even be misleading if taken too seriously.
characteristics of the drug product.The studies could include testing to assess the effect of high The combined results for the set of 11 typical
temperature,humidity,oxidation,photolysis and/or thermal cycling. stress conditions are shown in the summary dia-
gram (see Figure 1). The extraordinary observa-
54 Pharmaceutical Technology FEBRUARY 2005 www.phar mtech.com
Data and Review
tion is that only in a rather small fraction of cases does ad-
equate degradation occur with the applied typical stress con-
ditions. The results were combined in one chart because gen-
erally the same pie-chart distribution was obtained for each
individual stress condition. It was frequently observed that
although one stress condition was too soft for one compound,
it led to massive degradation for another compound. Appar-
ently, it is still difficult to generate the relevant degradation
products with a fixed set of stress conditions. Results indi-
cate that some fine-tuning of applied stress conditions or
stress duration will be necessary and that no universal set of
stress conditions exists. The large proportion of cases in the
false category is of particular concern because such stud-
ies may give a false idea of achieving relevant degradation.
On the basis of the degree of degradation, one can assume
that typically the right amount of stress was applied and, there-
fore, relevant degradation products could be expected. This
was not the case, however. The most probable cause for the
high percentage of irrelevant degradation may be the signifi-
cant difference in the thermodynamics of the applied stress
conditions, resulting in degradation pathways other than what
is observed during shelf-life stability-testing conditions. Ac-
cording to the IPG, these results demonstrate that degradation
from fast stress-testing conditions may be misleading and there-
fore should be interpreted with precaution.
Results from the screening test show that the typically ap-
plied conditions usually generate a lot of irrelevant degrada-
tion products, thereby leading to complicated results and a
potential for not producing relevant degradation products. In
addition, evaluating large numbers of irrelevant degradation
products may generate high amounts of workload. The screen-
ing study by the IPG, therefore, demonstrates the limited value
of using fast and severe stress-testing conditions. Our hypoth-
esis is that for successful stress testing, mild conditions should
be used that may result in storage times that are longer than in
usual stress testing.
Strategy toward a generic approach
The IPG tried to elaborate an applicable generic approach for
stress testing to produce representative samples for developing
stability-indicating methods for drug substances and drug prod-
ucts. The initial question was how much stress could be regarded
as adequate to generate such samples. The IPG group agreed
that the amount of stress applied to a sample should be selected
according to what is necessary to achieve purposeful degrada-
tion. An optimal degradation pattern generated during stress
testing would show only those degradation products observed
at the end of shelf life in regulatory stability studies and those
that might appear if the drug substance or drug product is not
handled or packed properly (see sidebar, Requirements for rel-
evant stress conditions).
Chromatograms thus obtained will be representative and not
too complicated to evaluate, which may be the case if drastic
conditions are applied and many second- and third-generation
degradation products are formed.
Stress testing should induce not more than 515% degrada-
tion of the main compound. A stress test should be stopped
56 Pharmaceutical Technology FEBRUARY 2005
Table I: Investigated set of fixed stress conditions.
Temperature Temperature Acid/base/
and moisture Light oxidative
30 min, 1 week 1 h xenon light 2 h 1 M HCl
121 C 70 C, ambient (7090 klx)
2 weeks 2 h xenon light 2 h NaOH
70C, ambient (7090 klx)
1 week 35 h UV light 2 h 3% H2O2
70 C, 100% RH (;210 W h/m2)
2 weeks
70 C, 100% RH
Table II: Type of degradation observed with a fixed set of
fast and severe stress conditions.
Category Explanation
Soft No significant degradation and therefore
no relevant degradation products observed
False Fair amount of degradation (,15%), however
no relevant degradation product(s) observed
Adequate Fair amount of degradation (,15%) and at least
one or all relevant degradation product(s) observed
Out of Between 15 and 100% degradation and at least
proportion one relevant degradation product observed
Overdone Between 15 and 100% degradation, however, no
relevant degradation products are observed
Requirements for relevant stress conditions
Should lead to the degradation of the main compound,but not more than 515%
Should lead to a good predictability of degradation pathways (i.e., a low
probability of drasticor falsedegradation)
Should be conducted for no longer than three months
when this percentage of degradation is achieved. It is not de-
sirable to generate samples with extensive degradation because
of their limited relevance and the formation of secondary degra-
dation products, which would lead to complicated degradation
patterns.
One issue to address is how long a stress test should be con-
tinued if the target is not achieved. The IPG proposes a maxi-
mum of 14 days for stress testing in solution (a maximum of
24 h for oxidative tests) to provide relevant stressed samples for
methods development. The group also proposes a three-month
period for drug substances in the solid state and drug products,
which may be too long for preformulation during early devel-
opment (e.g., early development screening or salt screening).
In such cases, more drastic conditions can be applied to gener-
ate results on a shorter course but at a risk that the degradation
patterns may not be predictive. In general, to evaluate their rel-
evance, results from stress tests should be compared with re-
sults obtained from long-term and accelerated stability studies
as soon as they are available.
The IPG developed a generic protocol for stress testing on
the basis of the experience and scientific expertise of its partic-
ipating companies and by taking into consideration the exist-
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Data and Review
ing regulatory guidance documents. The proposed conditions
should be sufficient and should normally induce realistic, pre-
dictive degradation. Predominant degradation products ob-
tained under these conditions should be regarded as relevant
for developing a stability-indicating method and will provide
valuable information about relevant degradation pathways. For
this purpose, it is essential to identify the observed degradation
products by structure elucidation, especially if they are formed
in more than one of the experiments included in the generic
protocol. However, common sense should be used when select-
ing degradation products for identification from a complicated
pattern. Identification work should not be mandatory unless a
particular degradation product has been shown to be relevant
under long-term or accelerated storage conditions.
Finally, the IPG decided that the agreed generic protocol for
stress testing should be developed using examples from the par-
ticipating companies, with evaluation according to both histor-
ical data from marketed products and, in some cases, results of
new experiments.
Program for stress testing drug substances in the solid state and
drug product. The IPG established conditions for stress testing
drug substances in the solid state and drug product (see Table
III). These conditions are regarded as sufficient to expose a solid-
state sample to temperature, humidity, and oxidation (oxida-
tion by open storage) stress. Testing periods are upper limits
and studies should be stopped when 515% degradation is
achieved.
Temperatures higher than those proposed in Table III (e.g.,
7090 8C) can be applied to rapidly generate method develop-
ment or preformulation samples. This approach, however, may
lead to various degradation kinetics and irrelevant degradation
products, as demonstrated in the screening experiment. Shorter
storage times should be avoided to maintain realistic thermo-
dynamics. Longer storage times are not recommended because
such samples are hardly available early in development and
should be considered only if no degradation is achieved after
three months.
During the evaluation of the generic stress program, a revi-
sion of ICH Q3B16 was published, in which light, heat, humid-
ity, acidbase hydrolysis, and oxidation are mentioned as stress
conditions to demonstrate the specificity of a stability indicat-
ing method for drug product. Light, heat, humidity and oxida-
tion (open storage) are considered to be covered by the condi-
tions listed in Table III. However, acidbase hydrolysis of the
drug product is considered as a less relevant stress condition by
the IPG members, provided that the information about the
degradation of the drug substance obtained under acidic and
basic conditions is taken into consideration.
Program for stress testing of drug substance in solution. Condi-
tions for stress testing of drug substance in solution are shown
in Table IV. For many final products, especially most solid oral
dosage forms, testing the drug substance in solution may be of
limited relevance. However, stress testing in solution is gener-
ally seen as relevant both for elucidation of degradation path-
ways and for specificity testing of the analytical method.
Stress testing in solution or in suspension of the drug sub-
stance often is conducted at temperatures above ambient (3,5).
58 Pharmaceutical Technology FEBRUARY 2005
Table III: Storage conditions for stress testing of drug
substance in solid state and drug product.
Storage conditions Testing period*
40 8C, 75 % RH; open storage** 3 months
5060 8C, ambient RH; open storage 3 months
Photostability; according to ICH according to ICH(67)
*3 months or 515% degradation, whatever comes first.
**For drug substances, typically a thin layer of material is spread in
a petri dish. Open storage is recommended if possible.
Table IV: Conditions for stress testing of drug substance
in solution.
Storage conditions Testing period*
pH 62, room temperature 2 weeks
pH 67, room temperature 2 weeks
pH 1012, room temperature 2 weeks
H2O2, 0.12% at neutral pH, 24 h
room temperature
*Storage times given or 515% degradation, whatever comes first.
Such an approach increases the probability of generating irrel-
evant degradation products, especially secondary degradation
products, which was confirmed in the IPGs screening study.
After extensive discussion, the IPG proposed not to use elevated
temperatures as a means to accelerate degradation.
In special cases, however, testing the drug substance at ele-
vated temperature in solution may be of interest (e.g., to pre-
dict stability during autoclaving of a solution). Therefore, test-
ing a drug substance in solution under elevated temperatures
should be considered on a case-by-case basis.
Stress testing in solution should be conducted on dissolved
samples. Additives may be used to enhance the solubility of
compounds with limited aqueous solubility (e.g., cosolvents or
cyclodextrins). Caution should be taken in these cases because
several additives are not inert agents. Cyclodextrins, for instance,
may protect potentially reactive moieties in a molecular struc-
ture, whereas cosolvents may affect degradation mechanisms
and even induce degradation (e.g., formation of reaction prod-
ucts such as methyl or ethyl esters).
Suspensions are not recommended because degradation
might be influenced by the presence of particles, and degrada-
tion-sensitive moieties can be protected if a suspension is used.
Suspensions may sometimes be justified, however, especially if
the formulation is a suspension. The three pH regions given in
Table IV may be chosen with flexibility, depending on the pH
of the potential formulation, anticipated degradation, solubil-
ity, and so forth. Again, not more than 515 % degradation is
the target value. If this amount of degradation is not achieved,
the samples may be stored for a longer period of time or at
higher temperatures on a case-by-case basis.
Experiments should be performed in the laboratory without
protection from daylight. Exposure of solutions in photostabil-
ity testing chambers will normally lead to much degradation
and an overestimation of photosensitivity. If degradation oc-
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Data and Review

Table V: Retrospective evaluation of the generic stress-testing protocol.

Degradation products observed
Company A Company B Company C Company D
#1 #2 #3 Remark #1 Remark #1 Remark #1 #2 Remark
Drug substance in solid state
40 8C, 75 % RH, 3 months open storage N
50 8C,ambient RH, 3 months open storage N NP NP
Photostability, according ICH L **
Drug substance in solution
pH 1, 3 days, room temperature *
pH 2, 2 weeks, room temperature N H
pH 7, 2 weeks, room temperature N 3 days H
pH 10, 2 weeks, room temperature 6h
pH 11, 2 weeks, room temperature N
pH 12, 2 weeks, room temperature **
0.1 % H2O2, 24 h, room temperature H, L
1 % H2O2, 24 h room temperature H
2 % H2O2, 24 h, room temperature H, L **
Fe2+ (0.05 mM), 2 weeks, room temperature. N
Cu2+ (0.05 mM), 2 weeks, room temperature L
Drug product
40 8C, 75 % RH, 3 months open storage NP NP NP NP NP NP
5060 8C, ambient RH, NP NP NP NP NP NP NP
3 months open storage
Photostability, according to ICH NP NP NP
Real-time stability studies of drug product
25 8C, 60 % RH 18 months 36 months 24 months 5 years
30 8C, 70 % RH 3 years
40 8C, 75 % RH 6 months 3 months 3 months
40 8C, 80 % RH 18 months
50 8C, uncontrolled RH 18 months

*pH 1 was chosen for harsher stress conditions because Compound 1 is stable in acidic media.
**A known degradation product is formed under these stress conditions. This degradation product, however, is not formed during the real
time stability studies. Key: denotes observed; denotes not observed; NP denotes.not performed; H denotes additional degradation
products observed at higher levels than the relevant degradation products mentioned in the table; L denotes additional degradation products
observed at lower levels than the relevant degradation products mentioned in the table; N denotes no significant degradation. observed.

curs in samples exposed to ambient light, a dark control may Results

be useful to distinguish between photodegradation and other
mechanisms.
To test susceptibility to oxidation, 0.12 % of H2O2 at neu-
tral pH is the most predictive. A non-neutral pH may be help-
ful in cases of limited solubility or to distinguish between ox-
idative and other degradation.
Optional stress conditions for drug substance in solution. Routine
testing of radical initiators or transition metals such as Fe31 and
Cu21 as initiators or catalysts for oxidative degradation are not
generally regarded as relevant, and a case-by-case approach is
recommended. This means that the influence of radical initia-
tors or metal ions should be tested if expected to be relevant
(e.g., when these constituents are expected to be present in the
potential formulation or if a special susceptibility can be antic-
ipated). Testing Na2S2O3 as a reducing agent may be included
in stress testing studies in special cases if considered relevant.
60 Pharmaceutical Technology FEBRUARY 2005
The proposed stress testing conditions of the generic approach
were evaluated retrospectively by comparing the degradation
patterns obtained during real-time storage in regulatory stabil-
ity studies of marketed products with the degradation patterns
obtained by applying the conditions proposed by the generic
protocol. Four products developed by their respective manu-
facturers (companies A, B, C, and D) were compared (see Table
V). These compounds represent a range of chemical character-
istics that to some extent can be considered to be representa-
tive of low molecular weight compounds in the pharmaceuti-
cal industry.
The selected test compound from Company A had under-
gone previous regulatory stability studies and all of its stability
data, including degradation pathways, were known and avail-
able. Results indicated that all degradation products that were
observed in the regulatory stability study were readily detected
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Data and Review
under the stress conditions of the pro-
posed generic stress testing protocol (see
Table V). Degradation products 1, 2, and
3 were relevant. Although some irrelevant
degradation products were observed, for
example under the oxidative conditions,
the number of degradation products was
still easy to handle.
The test compound from Company B
is registered in most countries and also
62 Pharmaceutical Technology FEBRUARY 2005
described in a monograph in the European
Pharmacopoeia. The test compound is sus-
ceptible to oxidation, and the resulting ox-
idation product is the only relevant degra-
dation product for drug product stability.
It is also included as a named degradation
product in the pharmacopeial monograph
for the drug substance. Results show that
the relevant degradation product is read-
ily formed in the drug substance by sev-
eral tests in the generic stress-testing pro-
tocol (see Table V). The most relevant con-
dition was the forced photostability study,
which is required by ICH Q1B (12).
Degradation product 1 was also generated
in the presence of copper ions and by hy-
drogen peroxide. Under these conditions
the degradation product was not as clearly
dominating as in photostability testing,
and in the case of hydrogen peroxide, it
was not even the largest degradation prod-
uct. The oxidation of this compound is a
well-known feature of the substance class,
and hence, the degradation product would
be considered as a likely degradation prod-
uct, even without performing any stress
testing.
The drug product of this compound
(extended-release tablets) was not tested
according to the generic stress-testing pro-
tocol. A review of older data indicated that
the proposed conditions would not nec-
essarily generate the degradation product.
In particular, the tablet coating provides
an effective protection against photochem-
ical degradation. However, the degrada-
tion product was formed in the drug sub-
stance upon stress testing, and the
oxidation is well-known for the substance
class. Hence, the degradation product has
been considered as a most likely degrada-
tion product from early development of
this product and onward.
A marketed product from Company C
was selected as a test compound. Several
stability studies have been performed on
this compound, and the degradation
pathways are known. For the drug sub-
stance studies, degradation product 1,
which is not formed in the crystalline
drug substance during long-term and ac-
celerated storage, is formed by stress test-
ing in solution (neutral and alkaline pH),
as described in the generic stress-testing
protocol.
For drug product studies, degradation
product 1 is formed under the influence
of humidity and even under long-term
conditions when the ratio of active to ex-
cipient is worse (data for another dosage
strength were not given). In this example,
the proposed stress humidity conditions
were sufficient to induce degradation.
Company D also chose a well-docu-
mented compound with an extensively in-
vestigated degradation mechanism. Two
main degradation products are known for
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Data and Review
the various strengths of the marketed prod-
uct. Degradation product 1 is observed dur-
ing all long-term stability studies. This
degradation product is formed during 3
months open storage at 40 8C and 75% RH
of the drug substance, as well as in solution
at pH 7. Degradation product 2 is ob-
served during the long-term stability stud-
ies at higher temperatures (30 and 40 8C).
This degradation product is formed under
64 Pharmaceutical Technology FEBRUARY 2005
stress conditions in solution at acidic pH.
From these results, one can conclude that
both degradation products observed dur-
ing long-term stability studies were ob-
served with the drug substance in solu-
tion during the generic stress-testing
protocol.
Results demonstrated that all relevant
degradation products observed during
real-time stability studies of these four dif-
ferent drug products were formed during
the proposed generic stress-testing proto-
col (see Table V). In general, purposeful
degradation that is relevant to the prod-
ucts stability was obtained. The generated
samples were suitable for the development
of stability-indicating methods.
Conclusion
Results from stress testing studies are used
to assess drug substance and drug prod-
uct stability, to provide information about
possible degradation pathways, and to
demonstrate the stability-indicating ca-
pability of the analytical methods used.
Drastic conditions tend to generate irrel-
evant degradation in the sample and may
lead to methods optimized for the sepa-
ration of an impurity profile, which is not
relevant for an end-of-shelf-life sample.
The IPG developed a generic approach
for stress testing of drug substances and
drug products. Efforts were aimed to pre-
vent too-drastic conditions and to achieve
purposeful degradation, predictive for
what is relevant under long-term and ac-
celerated storage conditions. The proposed
stress testing program, which is in line with
requirements from the regulatory agencies,
was applied to four products developed by
the participating companies and evaluated
both retrospectively by historic data and by
new experiments. Results demonstrated that
the proposed generic approach is reason-
able and generates relevant, generally pre-
dictive results for the development of a sta-
bility-indicating method. Some of the
conditions included, however, still induce
irrelevant degradation.
Stress tests for developing a stability-
indicating method should always be de-
signed and evaluated with common sense
and chemical knowledge, keeping in mind
the manufacturing process and the nature
of the final drug product. The proposed
generic approach can be used as a start-
ing point to set up a stress testing study,
but a case-by-case approach for stress test-
ing is essential to allow flexibility. This is
also recognized by the regulatory agencies
because very detailed instructions about
how to perform stress testing are not given
in the available guidance documents.
A general experience in the IPG was
that the formal stability study under ac-
celerated conditions is highly predictive
for long-term storage and the impurity
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Data and Review
profile at the end of shelf life. These results should be used as
soon as they become available to verify the relevance of the stress
testing performed.
Acknowledgment
We wish to thank Jenny Ottosson for performing the stress test-
ing experiments; Magnus Erickson (AstraZeneca R&D, Ml-
ndal, Sweden) and Han Op't Land, Henritte Hamstra, and Piet
Hoogkamer (all at Solvay Pharmaceuticals B.V., The Nether-
lands) for their valuable comments on the manuscript; Laurent
Duhau (Aventis, France) for the inspiring discussions; Mark
Vermunt and Gerben Wynia for performing the screening study
experiments; and Eddy Ruyter (Akzo Nobel, N.V. Organon, The
Netherlands) for his comments.
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