Forced Degradation Study or Stress Testing- Dr. A. Amsavel Ph.D.pdf

Forced degradation Study/
Stress Testing of API and Pharmaceutical products

Scope & Purpose of
Forced Degradation Study / Stress Testing,
Stress condition - Principle and Theory,
Chemical degradation,
Testing of Samples,
Evaluation of Stress testing,
Stability Indicating Method
Conclusion/Outcome of the study
RDC 53/2015 -
Identification of degradation of impurity, degradation pathway, Specification for degradation impurity, Identification and qualification of degradants.
Effect of temperatures, Humidity
Acid , base hydrolysis, Oxidation
Metal Ion
Photolysis etc,
stability-indicating analytical method

1. 1
Forced degradation Study/
Stress Testing of API and
Pharmaceutical products
Dr. A. Amsavel M.Sc., B.Ed., Ph.D.,

2. An Over View
Introduction
Scope & Purpose
Forced Degradation Study / Stress Testing
Stress condition -Principle and Theory
Chemical degradation
Testing of Samples
Evaluation of Stress testing
Conclusion/Outcome of the study
ANVISA resolution RDC 53/2015- Forced degradation
legislative

3. Definition
Stress testing (drug substance)
Studies undertaken to elucidate the intrinsic stability of the
drug substance. Such testing is part of the development
strategy and is normally carried out under more severe
conditions than those used for accelerated testing.
Accelerated testing
Studies designed to increase the rate of chemical degradation
or physical changeof a drug substance or drug product by
using exaggerated storage conditions as part of the formal
stability studies.

4. Stability Indicating Method (SIM)
Without analytical methods it is not possible to know what has
happened during stability
Assay
Impurities/Degradation Products
Chiral Purity
Dissolution
Preservative Content
Use preliminary method to analyze stress stability studies
–evaluate ability of method to separate degradantsi.ethe specificity -confirm
peak purity, mass balance
Use this information to modify/improve method
Stability studies and analytical method development work together
4

5. Stress Testing / ForcedDegradation Study
Stress testing is also called as Forced Degradation Study.
Stability testing under conditions exceeding those used
for accelerated testing.
Stress testing is conducted on Drug molecule to discover
Potential degradation products,
Molecule's intrinsic stability
Degradation pathways by which it degrades,
Accuracy of analytical techniques employed to stability study
(to establish Stability Indicating Method –SIM)

6. Scope and Purpose
“To provide evidence of how the quality of an API or FPP varies with
time under the influence of a variety of environmental factors such
as temperature, humidity and light etc“
Understanding of the molecule behaviour under extreme conditions
To know the degradation impurities / path ways of degradation
Validation of analytical methods
To establish stability Indicating method
It is a tool to predict the stability problems-select LT/AT condition
To Select Packaging material
To establish storage conditions.
To establish/ Justify specification limits for degradation impurities

7. Use of Stress Study
The stress study is vital and this information can be used,
to generate a degradation product and to facilitate stability studies
to use failure investigation-degradation path & degradation impurity
structure
to develop and validate a stability-indicating analytical method
to establish stable formulations
to facilitate improvements in the manufacturing process and formulations
to distinguish degradation impurities /products in formulations that are
related to drug substances or non-drug substances (e.g., excipients)
to solve stability-related problems (Eg, Peak purity , mass balance)
to choose the correct storage conditions, appropriate packagin g

8. Stress Testing
Use one batch of API (pilot scale, or production
batch) for FDS
Should include:
Effect of temperatures
Humidity
Acid hydrolysis
Base hydrolysis
Oxidation
Metal Ion
Photolysis (generally as per Q1B)

9. Stress Testing
Purposeful degradation of Drug expose with acid, base, heat, light, or
oxidants to make 10-20% degradation. Greater than 10 -20% could
result in secondary degradants
10-30% loss of API assay (FDC guideline, as well as Generic
guideline)
between 5% to 20% degradation (some literature)
approximately 10 % degradation is optimal for method validation
(as assay limits are normally 90-110%)

balance between "purposeful degradation" and irrelevant
artifacts(e.g., secondary degradation products).
degradation products not formed during accelerated or long-
term stability studies-need not always to be examined

10. Stress Testing
The study of the forced degradation profile must comply with the
following requirements as per RDC 58/2015:
Conducting the study on a commercial batch, on a laboratory, pilot or
industrial scale of the drug
study can be carried out with the formulation, with the placebo and
with the API isolated
The study of the forced degradation profile must be carried out in all
concentrations of the drug.
In the case of fixed dose combinations, forced degradation studies
must also be carried out with the API, associated and in the
formulation.

11. Stress Testing –Flow

12. Stress Testing - Matrix
Recommended Stress Conditions Of API & Drug Products
Stress
Condition
Examples
API Drug Products
Solid
Solution /
Suspension
Solid
Solution /
Suspension
Acid/ Base 0.01 to 0.1N – X – X
Oxidative
0.3% H
2
O
2 /
Metal ion – X – X
Light 2 millionLux hrs X X X X
Temperature50ºC to 70ºC X X X X
Humidity
50ºC to 70ºC and
60 to 90%RH.
X – X –

13. Chemical Degradation
Chemical degradation of DS / DP occurs by several
pathways like -Elevated temperature, hydrolysis,
oxidation, photolysis, racemization.
Degradation might have the following impact,
lower drug content will lower of therapeutic effect,
Formation of toxic degradation product,
decrease bioavailability etc.

14. Thermal -Stress Testing
Thermal stress testing condition the effect of
temperature is determined by the following
Arrhenius equation,
k -Specific reaction rate / rate constant
A-frequency factor
Ea-Energy of activation,
R-Gas constant (1.987 cal/degmole)
T-absolute temperature

15. Effect of DS / DP at different pH
Hydrolysis degradation is a common chemical reaction at different
pH.A functional group in a drug substance (DS) molecule may
hydrolyze and may form degradation.
To understand the hydrolysis character of DS, perform the study by
adding Acid or Base or buffer solutions at extreme pH,
Decide if the molecule will survive passage through the stomach
Assess the enteric coating necessary
should the drug be given by injection

16. Acid /Base Hydrolysis-Principle
Should study the FPP containing water such as emulsion ,
suspension , solutions , etc.
Drugs affected due to hydrolysis by hydrogen ion (acid) or
hydroxyl ion (base).
Drugs which are affected by moisture (higher %RH) from
atmosphere.
Drug is decomposed with solvent eg. Ethyl alcohol or
polyethylene glycol
Drugs that may undergo hydrolysis are the Esters, Amide, Alkali,
Acid etc.

17. Hydrolysis Reaction
Ester Hydrolysis: cleavage reaction gives acyl & acid.
RCOOR’ (ester) + H2O RCOOH (acid) + HOR’(alcohol)
EgAspirin, Atropine, Physostigmine, procaine etc
Amide Hydrolysis: Amide is more stable than ester, susceptible to
acid /base hydrolysis. Reaction is cleavage of amide gives an Amine
and Acid .
RCONHR’(amide) + H
2O RCOOH + NH2R’ (amine)
Eg: chloramphenicol, barbiturates .
Lactonesalso undergo hydrolysis

18. Oxidation-Theory
Oxidation is the loss of electrons while reduction is the gain of
electrons.
Oxidation of drugs is possible due by environment i.e, light, trace
elements, oxygen and oxidizing agent
exposed to atmospheric oxygen.
addition of oxygen or removal of hydrogen.
Reaction of functional group
-2ºand 3ºAmines to N-oxide, hydroxylamine by oxidation
-Aldehyde to Acid by oxidation

19. Auto-oxidation
Auto-oxidation isthe spontaneous oxidation.
The reaction between the compound and molecular oxygen,
which initiating the chain reaction is called auto-oxidation.
Free radicals produced during initial reaction are highly reactive
and further catalyzethe reaction produced additional free
radicals and causing a chain reaction.
Heavy metals such as copper, iron, cobalt , and nickel have been
known to catalyzethe oxidative degradation.
Heat and light further influence the kinetics of oxidative
degradation

20. Oxidation Reaction
Initiation: Formation of free radicals is taken place .
R-H R. + [H’}
Propagation: Free radical is regenerated and react with more
oxygen .
R. + O
2 RO2
RO2+ R’H ROOH + R’.
Hydrogen peroxide Decomposition:
ROOH RO + OH
Termination: free radicals react with each other resulting inactive
R’-O
2+ X Inactive product
RO
2+ R’O2 Inactive product

21. Photolysis-Theory
In order to confirm that exposure to Light (ultraviolet or fluorescing) does
not cause an excessively undesirable change.
When molecules are exposed to electromagnetic radiation they absorb
light (photons) at specific wavelength by increase in energy which can
Cause decomposition.
Absorbed Energy may retained or transferred
converted to heat
light emission at a new wavelength (fluorescence, phosphorescence).
Natural sun light lies in wavelength range (290–780nm). Higher energy
(UV) range (290 -320 nm) photons hit molecule and cause photo-
degradation. Common used light is at λ300 to 800 nm
Protect from light, in case of degradation
API -Store the product in dark or Black bags for APIs,
FPP-coating , Opaque polymer films, amber coloredbottles

22. Photolysis Condition & Reaction
Photolytic degradation is by oxidized or non-oxidizing through a free
radical reaction. Functional group like carbonyls, nitro aromatic N-oxides,
alkenes, aryl chlorides, weak C -H and O-H bonds, and sulfide etc.
The oxidative process involves isomerization, dimerization, etc.
Oxidation reaction eg
C-X hetero bonds breaks down by homolysis.
C-S bonds breaks down by deamination.
Photolytic study performed by expose the sample to 200 watts/m
2
for DS
or 1.2 watts/sq. inch for DP.
2 -6max million lux hour is suggested for study.

23. Conducting Stress Study
Selecting Concentration for Stress study
The precise concentration stress testing is not mention in any of the
regulatory guidelines.
However, initiate the study with 1 mg/ml as an initial concentration
considering detection of low level degradation impurities.
Based on the above increase or decrease the concentration to get 10-
20% degradation.
Start at room temperature and increases to higher temperature (50 to
70 °C at 10° C interval). Time period is 1 day to 7 days maximum.
After exposure stop the reaction, cool to RT and neutralized with a
suitable buffer or acid or base and test
Resulting sample is analyzedby measuring loss of parent drug .

24. Conducting Stress Study
Condition of Hydrolytic studies:
0.1M to 1M Aqueous acid or base is preferable for hydrolysis studies.
Sodium hydroxide or Potassium hydroxide solution is acceptable for
Base hydrolysis,
Hydrochloric acid or Sulfuricacid is acceptable reagents for Acid
hydrolysis.
If water is not suitable for solubility of drug molecule than co-solvent
can be added and can be dissolved in acid solution(HCl ) or base
solution(NaOH).
Start at room temperature and increases to higher temperature (50 to
70 °C at 10° C interval). Time period is 1 day to 7 days maximum.

25. Conducting Stress Study
Condition of Oxidation:
Preferably Use 0.1% to 3% H
2O
2(at pH level is neutral and at room level
temperature) for maximum of 7 days or at 40° C for 1 to 7 days (at room
level temperature) up to degradation of 20% maximum.
Other oxidizing agents like metal ions, oxygen and radical initiators also
can be used (e.g., Azobisisole, AIBN, etc.)
Condition of Humidity :
Humidity plays a significant role in degradation. Perform > 75% RH and
< 90% RH for 1 week

26. Conducting Stress Study
Photolytic Degradation Condition:
In order to confirm that exposure to Light does not cause an excessively
undesirable change.
The main degrades of the pharmacological substance are formed by
photo stability testing when exposed to ultraviolet (UV) or fluorescing
(fluorescing) light.
For more detail -Refer Stability Testing: PhotostabilityTesting Of New
Drug Substances And Products ICH Q1b

27. Recommended Conditions for DS (API)
Type of Study Conditions Time Extreme Conditions
Acid Hydrolysis 0.1 –1N HClat 70
o
C Few hours –7 days
1N HClat 70
o
C for 7
Days
Base Hydrolysis 0.1 –1N NaOHat 70
o
CFew hours –7 days
1N NaOH at 70
o
C for 7
Days
Thermal hydrolysis Aqueous solution 70
o
C Few hours –7 days 7 days / 70
o
C
Oxidative /Solutions
0.3 –3% H
2O
2at
ambient in the dark
Few hours –7 days 3% H
2O
2for 7 days
5mM Fe III or Cu II at
room temperature
Few hours –7 days 7 days
Thermal solid Exposure at 70
o
C Up to 3 weeks 3 weeks at 70
o
C
Thermal / Humidity 70
o
C &75%RH Up to 3 weeks 3 weeks at 70
o
C/75%RH
Photo-Degradation Fluorescent & UV light
* >2 times the ICH Q1B option 2 conditions
*> 4 times the ICH Q1B option 2 conditions
27
*ICH Q1B option 2 –at 5000 ± 200 lux white light and 2 ±0.2 watts/square meter. This means a complete test takes: ~10 days white
light (when set to 5kLux) & ~4 days UV light (when set to 2 watts/square meter). To achieve 1.2 million lux hours and 200 watt
hrs/square meter. A control sample is to be placed in the cabinet but is to be protected from light.

28. Recommended Conditions for Drug products
Type of Study Conditions Time Extreme Conditions
Acid Hydrolysis0.1 –1N HClat 70
o
C Few hours –7 days
1N HClat 70
o
C for 7
Days
Base Hydrolysis 0.1 –1N NaOHat 70
o
CFew hours –7 days
1N NaOH at 70
o
C for 7
Days
Thermal hydrolysis Aqueous solution 70
o
C Few hours –7 days7 days / 70
o
C
Oxidative /Solutions
0.3 –3% H
2O
2at ambient
in the dark
Few hours –7 days3% H
2O
2for 7 days
5mM Fe (III) or Cu (II )at
room temperature
Few hours –7 days7 days
Thermal solid
Exposure at 70
o
C, may vary
if oxidation is expected
Up to 3 weeks 3 weeks at 70
o
C
Thermal / Humidity 70
o
C &75%RHUp to 3 weeks 3 weeks at 70
o
C/75%RH
28
*ICH Q1B option 2 –at 5000 ±200 lux white light and 2 ±0.2 watts/square meter. This means a complete test takes: ~10 days white light
(when set to 5kLux) & ~4 days UV light (when set to 2 watts/square meter). To achieve 1.2 million lux hours and 200 watt hrs/square
meter. A control sample is to be placed in the cabinet but is to be protected from light.

29. How to Confirm Analytical Method
Analytical methods for identification of degradation products
The degradation products have to be identified and quantified by an analytical
method. If the degradation products are not identified by the selected analytical
method, the method does not fit for the intended use.
Ensure before validating the Analytical methods and it is stability indicating .
Establish the selectivity /specificity to assess separation of impurities and there is
no co-elution with DS.
Verification of the chromatographic purity of the peak of the API using photodiode
array (PDA) detection, to know the homogeneity of the spectral peak.
If there is no degradation, Assay of the DS should remain same
Ensure the Limit of Detection (LoD) and Limit of Quantification (LoQ) to the
qualification level.

30. 303030
When Should Stress Study repeated?
The following situation performing Stress test
Changes or inclusions in the ROS of API
Changes of excipient in the composition of the finished product, or
justify.
Ensure APIs of each manufacturer if different ROS

31. Conclusion and Reporting
Spec Limit : Value above which a degradation product must be reported in the
stability study(ies);
Qualification Limit: V alue above which a degradation product must be qualified.
Degradation profile: D escription of the results and analytical activities used in
the detection, identification, structural elucidation and quantitative determination
of the degradation products present in the DS and DP.
Chromatographic purity of the API peak: Proof that there is no interference
from excipients, impurities and degradation products in the DS Peak. Verification
of the chromatographic purity of the peak of the API.
Degradation products: Impurities resulting from chemical changes that arise
during the manufacture or storage of the drug.
Qualification of degradation products: Assessment of the biological safety of
an individual degradation product or a given degradation profile at a specified
level.

32. Conclusion and Reporting
Technical justification must be provided for not using any of the
conditions.
The tests must promote degradation greater than 10% (ten percent) and
less than that which would lead to complete degradation of the sample,
compromising the test.
Where degradation is less than 10% (ten percent), provide technical
justification.

33. 333333
Identification and Qualification
Identification and qualification of the degradation product: Thresholds
for degradation products according to resolution RDC 53/ 2015
Maximum Daily
Dose 1
Limits 2
Notification
Limits
<1g 0.1%
>1g 0.05%
Identification
Limits <1mg 1.0% or 5μg ATD, whichever is less
1mg-10mg 0.5% or 20μg ATD, whichever is less
>10mg-2g 0.2% or 2mg ATD, whichever is less
> 2g 0.10%
Qualification
Limits <10 mg 1.0% or 50μg ATD, whichever is less
10 mg-100 mg 0.5% or 200μg ATD, whichever is less
>100 mg - 2g 0.2% or 3mg ATD, whichever is less
>2g 0.15%
a Maximum amount of the active
pharmaceuticalingredient administered per day.
b Limits of the degradation products are expressed as the percentage of the active pharmaceutical ingredient or as the
total daily administration (TDA) of a degradation product
c Average Daily Dosage

34. 343434
Identification and Qualification
(RDC 53/2015)
1.Maximum quantity of API administered per day.
2.Limits of degradation products are expressed as the % of API or as the total
daily administration (ATD) of a degradation product.
3.The degradation product(s) with a percentage or corresponding value
above the established identification limits must have their chemical
structure identified and individual quantification carried out.
4.The degradation product(s) with a percentage or corresponding value
above the identification limits and below the qualification limits that present
in their chemical structure characteristics that lead to the classification of a
potentially toxic product must have their safety profile established through
the biological safety assessment.

35. 353535
Identification and Qualification
5.The degradation product(s) with a percentage or corresponding value above the
established qualification limits, when they present in their chemical structure
characteristics that lead to the classification of a potentially toxic product, must, in
addition to complying with the provisions of §4º, have their safety profile established
through the assessment of biological safety.
6.The safety profile expressed in §5 and §6 will be established for those products that
meet the provisions of art. 10 and may be determined through genotoxicity
assessment and general toxicity studies using validated methodology and in
accordance with specific guidelines for conducting non- clinical safety studies
necessary for drug development.

36. 363636
Identification and Qualification
The degradation product may be considered qualified when it meets at least
one of the following conditions :
the degradation product is a significant metabolite found during studies
in humans or animals
acceptance limit of a degradation product is justified scientifically /
official compendia
ensure appropriate limit is based on toxicity studies; or the exposure is
equal to or lower than that expressed in the list published (Included
byResolution 821/2023)
The acceptance limits for each individual degradation product and the
total limit of degradation products must be included in the drug release
specifications and the stability study.

37. 373737
Threshold & Qualification as per ICH Q3B
Thresholds for degradation products according to ICH Q3B

38. 383838
Benefits of Forced Degradation Study
Evaluate a inherent sensitivity of molecule and product's to various
variables and different combinations of variables, including
temperature, humidity, pH level, oxygen concentration, light, and the
presence of catalysts.
pathways and mechanisms for degradation
QbDknowledge space as it helps to understand the chemical reactivity
& Establishing mass balance early due to degradation
developing and validating methods to demonstrate stability
filter to quickly select formal long-term stability studies
distribution and storage.

39. 393939
Reference
Stability Testing of APIs and FPPs --WHO Stability Guide in TRS-953 (2009) Annex 2:
TRS 929 Annex 5- Guideline for registration of FDC (appendix 3,Table A.1)
ICH Q1A(R2), B, C, D & E : Guidance on Standard Stability Testing
The ICH Q3A(R2) Guidelines Described “Impurities in New Drug Substances”,
ANVISA resolution RDC 53; 08/12/2015- forced degradation legislative
Guideline on stability testing for applications for variations to a marketing
authorization (EMA 2011)
FDA-CFR 211.166.-
Forced degradation studies – comparison between ICH, EMA, FDA and WHO
guidelines and ANVISA’s resolution RDC 53/2015 -Helene Janzen -Master thesis -
Bonn 2016

40. 40
Thank You
Q&A