Pharmaceutical Technology: FDA Perspectives: Scientific Considerations of Page 1of 6
May 2, 2012
FDA Perspectives: Scientific Considerations of Forced
Degradation Studies in ANDA Submissions
By Ragine Maheswaran
The author outlines the scientific aspects of forced degradation studies that should be
considered in relation to ANDA submissions.
Forced degradation is synonymous with stress testing and purposeful degradation. Purposeful
degradation can be a useful tool to predict the stability of a drug substance or a drug product with effects
on purity, potency, and safety. It is imperative to know the impurity profile and behavior of a drug
substance under various stress conditions. Forced degradation also plays an important role in the
development of analytical methods, setting specifications, and design of formulations under the quality-by-design (QbD) paradigm. The nature of the stress testing depends on the individual drug substance
and the type of drug product (e.g., solid oral dosage, lyophilized powders, and liquid formulations)
involved (1).
The International Conference on Harmonization (ICH) Q1B guideline provides guidance for performing
photostability stress testing; however, there are no additional stress study recommendations in the ICH
stability or validation guidelines (2). There is also limited information on the details about the study of
oxidation and hydrolysis. The drug substance monographs of Analytical Profiles of Drug Substances and
Excipients provide some information with respect to different stress conditions of various drug
substances (3).
The forced degradation information provided in the abbreviated new drug application (ANDA)
submissions is often incomplete and in those cases deficiencies are cited. An overview of common
deficiencies cited throughout the chemistry, manufacturing, and controls (CMC) section of the ANDAs
has been published (4 –6). Some examples of commonly cited deficiencies related to forced degradation
studies include the following:
•Your drug substance does not show any degradation under any of the stress conditions. Please
repeat stress studies to obtain adequate degradation. If degradation is not achievable, please
provide your rationale.
•Please note that the conditions employed for stress study are too harsh and that most of your drug
substance has degraded. Please repeat your stress studies using milder conditions or shorter
exposure time to generate relevant degradation products.
•It is noted that you have analyzed your stressed samples as per the assay method conditions. For
the related substances method to be stability indicating, the stressed samples should be analyzed
using related substances method conditions.
•Please state the attempts you have made to ensure that all the impurities including the
degradation products of the unstressed and the stressed samples are captured by your analytical
method.
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Pharmaceutical Technology: FDA Perspectives: Scientific Considerations of Page 2of 6•Please provide a list summarizing the amount of degradation products (known and unknown) in
your stressed samples.
•Please verify the peak height requirement of your software for the peak purity determination.
•Please explain the mass imbalance of the stressed samples.
•Please identify the degradation products that are formed due to drug-excipient interactions.
•Your photostability study shows that the drug product is very sensitive to light. Please explain how
this is reflected in the analytical method, manufacturing process, product handling, etc.
In an attempt to minimize deficiencies in the ANDA submissions, some general recommendations to
conduct forced degradation studies, to report relevant information in the submission, and to utilize the
knowledge of forced degradation in developing stability indicating analytical methods, manufacturing
process, product handling, and storage are provided in this article.
Stress conditions
Typical stress tests include four main degradation mechanisms: heat, hydrolytic, oxidative, and
photolytic degradation. Selecting suitable reagents such as the concentration of acid, base, or oxidizing
agent and varying the conditions (e.g., temperature) and length of exposure can achieve the preferred
level of degradation. Over-stressing a sample may lead to the formation of secondary degradants that
would not be seen in formal shelf-life stability studies and under-stressing may not serve the purpose of
stress testing. Therefore, it is necessary to control the degradation to a desired level. A generic
approach for stress testing has been proposed to achieve purposeful degradation that is predictive of
long-term and accelerated storage conditions (7). The generally recommended degradation varies
between 5-20% degradation (7 –10). This range covers the generally permissible 10% degradation for
small molecule pharmaceutical drug products, for which the stability limit is 90%-110% of the label claim.
Although there are references in the literature that mention a wider recommended range (e.g., 10-30%),
the more extreme stress conditions often provide data that are confounded with secondary degradation
products.
Photostability. Photostability testing should be an integral part of stress testing, especially for photo-labile compounds. Some recommended conditions for photostability testing are described in ICH Q1B
Photostability Testing of New Drug Substances and Products (2). Samples of drug substance, and
solid/liquid drug product, should be exposed to a minimum of 1.2 million lux hours and 200 watt hours
per square meter light. The same samples should be exposed to both white and UV light. To minimize
the effect of temperature changes during exposure, temperature control may be necessary. The light-exposed samples should be analyzed for any changes in physical properties such as appearance,
clarity, color of solution, and for assay and degradants. The decision tree outlined in the ICH Q1B can be
used to determine the photo stability testing conditions for drug products. The product labeling should
reflect the appropriate storage conditions. It is also important to note that the labeling for generic drug
products should be concordant with that of the reference listed drug (RLD) and with United States
Pharmacopeia (USP) monograph recommendations, as applicable.
Heat. Thermal stress testing (e.g., dry heat and wet heat) should be more strenuous than recommended
ICH Q1A accelerated testing conditions. Samples of solid-state drug substances and drug products
should be exposed to dry and wet heat, whereas liquid drug products can be exposed to dry heat. It is
recommended that the effect of temperature be studied in 10 °C increments above that for routine
accelerated testing, and humidity at 75% relative humidity or greater (1). Studies may be conducted at
higher temperatures for a shorter period (10). Testing at multiple time points could provide information
on the rate of degradation and primary and secondary degradation products. In the event that the stress
conditions produce little or no degradation due to the stability of a drug molecule, one should ensure thatthe stress applied is in excess of the energy applied by accelerated conditions (40 °C for 6 months)
before terminating the stress study.
Acid and base hydrolysis. Acid and base hydrolytic stress testing can be carried out for drug
substances and drug products in solution at ambient temperature or at elevated temperatures. The
selection of the type and concentrations of an acid or a base depends on the stability of the drug
substance. A strategy for generating relevant stressed samples for hydrolysis is stated as subjecting the
drug substance solution to various pHs (e.g., 2, 7, 10 –12) at room temperature for two weeks or up to a
maximum of 15% degradation (7). Hydrochloric acid or sulfuric acid (0.1 M to 1 M) for acid hydrolysis
and sodium hydroxide or potassium hydroxide (0.1 M to 1 M) for base hydrolysis are suggested as
suitable reagents for hydrolysis (10). For lipophilic drugs, inert co-solvents may be used to solubilize the
drug substance. Attention should be given to the functional groups present in the drug molecule when
selecting a co-solvent. Prior knowledge of a compound can be useful in selecting the stress conditions.
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Pharmaceutical Technology: FDA Perspectives: Scientific Considerations of Page 3of 6For instance, if a compound contains ester functionality and is very labile to base hydrolysis, low
concentrations of a base can be used. Analysis of samples at various intervals can provide information
on the progress of degradation and help to distinguish primary degradants from secondary degradants.
Oxidation. Oxidative degradation can be complex. Although hydrogen peroxide is used predominantly
because it mimics possible presence of peroxides in excipients, other oxidizing agents such as metal
ions, oxygen, and radical initiators (e.g., azobisisobutyronitrile, AIBN) can also be used. Selection of an
oxidizing agent, its concentration, and conditions depends on the drug substance. Solutions of drug
substances and solid/liquid drug products can be subjected to oxidative degradation. It is reported that
subjecting the solutions to 0.1%-3% hydrogen peroxide at neutral pH and room temperature for seven
days or up to a maximum 20% degradation could potentially generate relevant degradation products
(10). Samples can be analyzed at different time intervals to determine the desired level of degradation.
Different stress conditions may generate the same or different degradants. The type and extent of
degradation depend on the functional groups of the drug molecule and the stress conditions.
Analysis method
The preferred method of analysis for a stability indicating assay is reverse-phase high-performance
liquid chromatography (HPLC). Reverse-phase HPLC is preferred for several reasons, such as its
compatibility with aqueous and organic solutions, high precision, sensitivity, and ability to detect polar
compounds. Separation of peaks can be carried out by selecting appropriate column type, column
temperature, and making adjustment to mobile phase pH. Poorly-retained, highly polar impurities should
be resolved from the solvent front. As part of method development, a gradient elution method with
varying mobile phase composition (very low organic composition to high organic composition) may be
carried out to capture early eluting highly polar compounds and highly retained nonpolar compounds.
Stressed samples can also be screened with the gradient method to assess potential elution pattern.
Sample solvent and mobile phase should be selected to afford compatibility with the drug substance,
potential impurities, and degradants. Stress sample preparation should mimic the sample preparation
outlined in the analytical procedure as closely as possible. Neutralization or dilution of samples may be
necessary for acid and base hydrolyzed samples. Chromatographic profiles of stressed samples should
be compared to those of relevant blanks (containing no active) and unstressed samples to determine the
origin of peaks. The blank peaks should be excluded from calculations. The amount of impurities (known
and unknown) obtained under each stress condition should be provided along with the chromatograms
(full scale and expanded scale showing all the peaks) of blanks, unstressed, and stressed samples.
Additionally, chiral drugs should be analyzed with chiral methods to establish stereochemical purity and
stability (11, 12).
The analytical method of choice should be sensitive enough to detect impurities at low levels (i.e., 0.05%
of the analyte of interest or lower), and the peak responses should fall within the range of detector's
linearity. The analytical method should be capable of capturing all the impurities formed during a formal
stability study at or below ICH threshold limits (13, 14). Degradation product identification and
characterization are to be performed based on formal stability results in accordance with ICH
requirements. Conventional methods (e.g., column chromatography) or hyphenated techniques (e.g., LC
–MS, LC –NMR) can be used in the identification and characterization of the degradation products. Use
of these techniques can provide better insight into the structure of the impurities that could add to the
knowledge space of potential structural alerts for genotoxicity and the control of such impurities with
tighter limits (12 –17). It should be noted that structural characterization of degradation products is
necessary for those impurities that are formed during formal shelf-life stability studies and are above the
qualification threshold limit (13).
Various detection types can be used to analyze stressed samples such as UV and mass spectroscopy.
The detector should contain 3D data capabilities such as diode array detectors or mass spectrometers to
be able to detect spectral non-homogeneity. Diode array detection also offers the possibility of checking
peak profile for multiple wavelengths. The limitation of diode array arises when the UV profiles are
similar for analyte peak and impurity or degradant peak and the noise level of the system is high to mask
the co-eluting impurities or degradants. Compounds of similar molecular weights and functional groups
such as diastereoisomers may exhibit similar UV profiles. In such cases, attempts must be made to
modify the chromatographic parameters to achieve necessary separation. An optimal wavelength should
be selected to detect and quantitate all the potential impurities and degradants. Use of more than one
wavelength may be necessary, if there is no overlap in the UV profile of an analyte and impurity or
degradant peaks. A valuable tool in method development is the overlay of separation signals at different
wavelengths to discover dissimilarities in peak profiles.
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Pharmaceutical Technology: FDA Perspectives: Scientific Considerations of Page 4of 6Peak purity analysis. Peak purity is used as an aid in stability indicating method development. The
spectral uniqueness of a compound is used to establish peak purity when co-eluting compounds are
present.
Peak purity or peak homogeneity of the peaks of interest of unstressed and stressed samples should be
established using spectral information from a diode array detector. When instrument software is used for
the determination of spectral purity of a peak, relevant parameters should be set up in accordance with
the manufacturer's guidance. Attention should be given to the peak height requirement for establishing
spectral purity. UV detection becomes non linear at higher absorbance values. Thresholds should be set
such that co-eluting peaks can be detected. Optimum location of reference spectra should also be
selected. The ability of the software to automatically correct spectra for continuously changing solvent
background in gradient separations should be ascertained.
Establishing peak purity is not an absolute proof that the peak is pure and that there is no co-elution with
the peak of interest. Limitations to peak purity arise when co-eluting peaks are spectrally similar, or
below the detection limit, or a peak has no chromophore, or when they are not resolved at all.
Mass balance. Mass balance establishes adequacy of a stability indicating method though it is not
achievable in all circumstances. It is performed by adding the assay value and the amounts of impurities
and degradants to evaluate the closeness to 100% of the initial value (unstressed assay value) with due
consideration of the margin of analytical error (1).
Some attempt should be made to establish a mass balance for all stressed samples. Mass imbalance
should be explored and an explanation should be provided. Varying responses of analyte and impurity
peaks due to differences in UV absorption should also be examined by the use of external standards.
Potential loss of volatile impurities, formation of non-UV absorbing compounds, formation of early
eluants, and potential retention of compounds in the column should be explored. Alternate detection
techniques such as RI LC/MS may be employed to account for non-UV absorbing degradants.
Termination of study
Stress testing could be terminated after ensuring adequate exposure to stress conditions. Typical
activation energy of drug substance molecules varies from 12 –24 kcal/mol (18). A compound may not
necessarily degrade under every single stress condition, and general guideline on exposure limit is cited
in a review article (10). In circumstances where some stable drugs do not show any degradation under
any of the stress conditions, specificity of an analytical method can be established by spiking the drug
substance or placebo with known impurities and establishing adequate separation.
Other considerations
Stress testing may not be necessary for drug substances and drug products that have pharmacopeial
methods and are used within the limitations outlined in USP <621>. In the case where a generic drug
product uses a different polymorphic form from the RLD, the drug substance should be subjected to
stress testing to evaluate the physiochemical changes of the polymorphic form because different
polymorphic forms may exhibit different stability characteristics.
Forced degradation in QbD paradigm
A systematic process of manufacturing quality drug products that meet the predefined targets for the
critical quality attributes (CQA) necessitates the use of knowledge obtained in forced degradation
studies.
A well-designed, forced degradation study is indispensable for analytical method development in a QbD
paradigm. It helps to establish the specificity of a stability indicating method and to predict potential
degradation products that could form during formal stability studies. Incorporating all potential impurities
in the analytical method and establishing the peak purity of the peaks of interest helps to avoid
unnecessary method re-development and revalidation.
Knowledge of chemical behavior of drug substances under various stress conditions can also provide
useful information regarding the selection of excipients for formulation development. Excipient
compatibility is an integral part of understanding potential formulation interactions during product
development and is a key part of product understanding. Degradation products due to drug-excipient
interaction or drug-drug interaction in combination products can be examined by stressing samples of
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Pharmaceutical Technology: FDA Perspectives: Scientific Considerations of Page 5of 6drug substance, drug product, and placebo separately and comparing the impurity profiles. Information
obtained regarding drug-related peaks and non-drug-related peaks can be used in the selection and
development of more stable formulations. For instance, if a drug substance is labile to oxidation, addition
of an antioxidant may be considered for the formulation. For drug substances that are labile to acid or
undergo stereochemical conversion in acidic medium, delayed-release formulations may be necessary.
Acid/base hydrolysis testing can also provide useful insight in the formulation of drug products that are
liquids or suspensions.
Knowledge gained in forced degradation studies can facilitate improvements in the manufacturing
process. If a photostability study shows a drug substance to be photolabile, caution should be taken
during the manufacturing process of the drug product. Useful information regarding process
development (e.g., wet versus dry processing, temperature selection) can be obtained from thermal
stress testing of drug substance and drug product.
Additionally, increased scientific understanding of degradation products and mechanisms may help to
determine the factors that could contribute to stability failures such as ambient temperature, humidity,
and light. Appropriate selection of packaging materials can be made to protect against such factors.
Conclusion
An appropriately-designed stress study meshes well with the QbD approaches currently being promoted
in the pharmaceutical industry. A well-designed stress study can provide insight in choosing the
appropriate formulation for a proposed product prior to intensive formulation development studies. A
thorough knowledge of degradation, including mechanistic understanding of potential degradation
pathways, is the basis of a QbD approach for analytical method development and is crucial in setting
acceptance criteria for shelf-life monitoring. Stress testing can provide useful insight into the selection of
physical form, stereo-chemical stability of a drug substance, packaging, and storage conditions. It is
important to perform stress testing for generic drugs due to allowable qualitative and quantitative
differences in formulation with respect to the RLD, selection of manufacturing process, processing
parameters, and packaging materials.
Acknowledgments
The author would like to thank Bob Iser, Naiqi Ya, Dave Skanchy, Bing Wu, and Ashley Jung for their
scientific input and support.
Ragine Maheswaran, PhD, is a CMC reviewer at the Office of Generic Drugs within the Office of
Pharmaceutical Science, under the US Food and Drug Administration's Center for Drug Evaluation and
Research, aran@ [aran@]
Disclaimer: The views and opinions in this article are only those of the author and do not necessarily
reflect the views or policies of the US Food and Drug Administration.
References
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2. ICH, Q1B Stability Testing: Photostability Testing of New Drug Substances and Products (Geneva,
Nov. 1996).
3. H. Brittain, Analytical Profiles of Drug Substances and Excipients (Academic Press, London, 2002).
4. A. Srinivasan and R. Iser, Pharm. Technol. 34 (1), 50
–59 (2010).
5. A. Srinivasan, R. Iser, and D. Gill, Pharm. Technol. 34(8), 45 –51 (2010).
6. A. Srinivasan, R. Iser, and D. Gill, Pharm. Technol. 35 (2), 58 –67 (2011).
7. S. Klick, et al., l. 29 (2) 48 –66 (2005).
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9. D. W. Reynolds, et al., l. 26 (2), 48 –56 (2002).
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Pharmaceutical Technology: FDA Perspectives: Scientific Considerations of Page 6of 611. FDA, Guidance for Industry on Analytical Procedures and methods Validation Chemistry,
Manufacturing, and Controls Documentation (draft) (Rockville, MD, Aug. 2000).
12. ICH, Q6A: Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and
New Drug Products: Chemical Substances (Geneva, Oct. 1999).
13. ICH, Q3A(R2) Impurities in New Drug Substances (Geneva, Oct. 2006).
14. ICH, Q3B(R2) Impurities in New Drug Products (Geneva, June 2006).
15. FDA, Guidance for Industry ANDAs: Impurities in Drug Substances (draft), (Rockville, MD, Aug.
2005).
16. FDA, Guidance for Industry ANDAs: Impurities in Drug Products (draft) (Rockville, MD, Nov. 2010).
17. EMA, Guideline on the Limits of Genotoxic Impurities, Committee for Medical Products for Human
Use (CHMP) (Doc. Ref EMA/CHMP/QWP/251344/2006) (Jan. 1, 2007).
18. K. A. Conners et al., Chemical Stability of Pharmaceuticals, Wiley and Sons, New York, New York,
2nd Ed., p. 19 (1986).
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