these slides discuss Impurity profiling Degradation characterization Stability testing & Accelerated stability testing (ICH) Evaluation of the test (shelf life) analytical method development ICH vs USP definition methods for identification method for the isolation of the impurity factors affecting the degradation of formulation What is degradation characterization general protocol of degradation conditions used for drug substance and drug product Degradation conditions Stress testing Container closure system

1. Submitted to :- Prof. Gita Chawla
Submitted by :- Shameer
Department :- Master of pharmacy (P.Analysis)
Sem/year :- 1sem/1year

2.  Impurity profiling
 Degradation characterization
 Stability testing & Accelerated stability testing
(ICH)
 Evaluation of the test (shelf life)

3.  Bulk drug industry forms base of all pharmaceutical
industries as it is the source of active
pharmaceutical ingredients (APIs) of specific quality
 The major challenge for both bulk drug industries
and pharmaceutical industries is to produce quality
products.
 Over the last few decades much attention is paid
towards the quality of pharmaceuticals that enter
the market.

To maintain and have a control over the quality and
purity of the pharmaceuticals products/ bulk drug

4.  Purity of active pharmaceutical ingredient
depends on several factors such as:-
Concept of purity
changes with time and
it is inseparable from
the developments I
analytical chemistry
The pharmacopoeias
specify not only purity
but also puts limits
which can be very
stringent on levels of
various impurities.

5.  Impurities in pharmaceuticals are unwanted chemicals
that remain with the Active Pharmaceutical Ingredients
(APIs) or develop during formulation or develop upon
ageing of both APIs and formulated APIs to medicines .
 The presence of these unwanted chemicals even in
small amounts may influence the efficacy and safety of
the pharmaceutical products
 Impurity profile is description of the identified and
unidentified impurities present in a typical batch of API
produced by a specific controlled production process

6.  New technology for manufacturing an existing drug it is essential to know the
structures of the impurities (by possessing the information synthetic organic
chemists are often able to change the reaction conditions in such a way that the
formation of the impurity can be avoided or its quantity reduced to an acceptable level)
 Having suggested structures for the impurities, they can be synthesized and
thus provide final evidence for their structures previously determined by
spectroscopic methods.
 The material synthesized can be used as an ‘impurity standard’ during
development of a selective method for the quantitative determination of the
impurity and the use of this method as part of the quality control testing of
every batch.
 In case of major impurities the synthesized or isolated material can be
subjected to toxicological studies thus greatly contributing to the safety of drug
therapy.
 For drug authorities the impurity profile of a drug substance is a good
fingerprint to indicate the level and constancy of the manufacturing process of
the bulk drug substance

9.  New drug development
requires meaningful and
reliable analytical data to
be produced at various
stages of the
development

11.  The key objective of this is to provide clarity to the overall life cycle,
qualification and governance of reference standards used in
development and control of new drugs.
 Reference standards serve as the basis of evaluation of both
process and product performance and are the benchmarks for
assessment of drug safety for patient consumption.
 These standards are needed, not only for the active ingredients in
dosage forms but also for impurities, degradation products, starting
materials, process intermediates, and excipients.

12.  These are the methods that are used routinely for
characterizing impurities.

13. These are the methods that are adopted for the separation

14.  It is often necessary to isolate
impurities.
 Generally, chromatographic and non-
chromatographic techniques are
used for isolation of impurities prior
its characterization
 The term ‘chromatographic reactor’
refers to the use of an analytical-
scale column as both a flow through
reactor, and simultaneously, as
separation medium for the reactant(s)

16.  Highly sophisticated instrumentation, such as MS
attached to a GC or HPLC, are inevitable tools in
the identification of minor components (drugs,
impurities, degradation products, metabolites) in
various matrices.
 Different techniques that are used for the
characterization of impurities are:-
NMR
MS
Hyphenated
methods

17.  The ability of NMR to provide information regarding the specific
bonding structure and stereochemistry of molecules of pharmaceutical
interest has made it a powerful analytical instrument for structural
elucidation.
 The ability of NMR- based diffusion coefficient determination to
distinguish between monomeric and dimeric substances was validated
using a standard mixture of authentic materials containing both
monomers and dimers
 Unfortunately, NMR has traditionally been used as a less sensitive
method compared to other analytical techniques. Conventional sample
requirements for NMR are on the order of 10 mg, as compared with MS,
which requires less than 1 mg.

18.  It has an increasingly significant impact on the pharmaceutical
development process over the past several decades.
 Advances in the design and efficiency of the interfaces, that directly
connect separation techniques with Mass Spectrometers have afforded
new opportunities for monitoring, characterizing, and quantification of
drug related substances in active pharmaceutical ingredients and
pharmaceutical formulations.
 If single method fails to provide the necessary selectivity, orthogonal
coupling of chromatographic techniques such as HPLC-TLC and HPLC-
CE, or coupling of chromatographic separations with information rich
spectroscopic methods such as HPLC-MS or HPLC-NMR may need to be
contemplated, but hopefully only as a development tool rather than a tool
for routine QC use.

19.  LC-MS-MS
 HPLC-DAD-MS
 HPLC-DAD-NMR-MS
 GC-MS
 LC-MS
A common goal for investigation of both
process and product degradation-related
impurities is to determine which of the
many potential impurities are, in fact,
produced in the manufacturing process
and which occur under a given set of
storage conditions.

20.  According to ICH guidelines on
impurities in new drug products,
identification of impurities below 0.1%
level, is not considered to be necessary,
unless potential impurities are expected
to be unusually potent or toxic.
According to ICH, the maximum daily
dose qualification threshold to be
considered is as follows as shown in
table no.2-5;< 2g/day 0.1 % or 1 mg per
day intake (whichever is lower) >2g/day

21.  It is describes as phenomena of
change in physiochemical properties
of any substance under study due to
various environmental factors or
interaction between the different
component of the formulation it can
lead to :-

22.  These are the products formed as a
result of the degradation of the
substance under study. These can be
sometimes just hydrolyzed or oxidized
products of actual substance whereas in
some cases degradation products can
be highly altered molecules on chemical
basis arising from the parent substance
molecules

23. Storage temperature
Humidity and moisture conditions
Photolytic degradation
pH of the formulation
Interaction between components
Microbial contamination
Interaction with the containers and closure system

24.  This involve complete profiling of the
degradation product formed in the
formulation
 Degradation products are impurities
in our formulation that arises due to
degradation of drug substances as
well as any excipients in the
formulation .so they are also
characterized as impurities and their
profiles are also maintained as per
impurity profiling guidelines

25.  Forced degradation studies are
carried out to achieve the following
purposes:
To establish degradation pathway of drug substances and drug products
To differentiate degradation products that are related to drug products
from those that are generally from the non-drug product in a formulation
To elucidate the structure of degradation products
To determine the intrinsic stability of a drug substance in the
formulation
To reveal the degradation mechanisms such as hydrolysis oxidation,
hemolysis or photolysis of the drug substance and drug products

27.  It is very important to know when to perform forced
degradation studies for the development of new drug
substance and new drug product.
 FDA guidance states that stress testing should be
performed in phase III of regulatory submission
process.
 Stress studies should be done in different pH
solutions, in the presence of oxygen and light, and at
elevated temperatures and humidity levels to
determine the stability of the drug substance.
 These stress studies are conducted on a single
batch. The results should be summarized and
submitted in an annual report
 An early stress study also gives timely
recommendations for making improvements in the
manufacturing process and proper selection of
stability-indicating analytical procedures

28.  Degradation of drug substances between 5% and
20% has been accepted as reasonable for
validation of chromatographic assays
 Some pharmaceutical scientists think 10%
degradation is optimal for use in analytical
validation for small pharmaceutical molecules for
which acceptable stability limits of 90% of label
claim is common
 No such limits for physiochemical changes, loss
of activity or degradation during shelf life have
been established for individual types or groups
of biological products
 Protocols for generation of product-related
degradation may differ for drug substance and
drug product due to differences in matrices and
concentrations. It is recommended that
maximum of 14 days for stress testing in
solution (a maximum of 24 h for oxidative tests)

29.  Frist of all keep in mind that Forced
degradation is carried out to produce
representative samples for developing
stability-indicating methods for drug
substances and drug products.
 The choice of stress conditions should
be consistent with the product's
decomposition under normal
manufacturing, storage, and use
conditions which are specific in each
case

30. • Photolytic
• Thermal
• Thermal
humidity
• Acid/base
• Oxidative
• Photolytic
• Thermal
• Thermal
humidity
• Oxidative
• Photolytic
• Thermal
• Thermal
humidity
• Photolytic
• Thermal
• Oxidative

31.  A minimal list of stress factors suggested for forced
degradation studies must include acid and base
hydrolysis, thermal degradation, photolysis,
oxidation and may include freeze- thaw cycles and
shear (There is no specification in regulatory guidelines about the
conditions of pH, temperature and specific oxidizing agents to be
used)
 The design of photolysis studies is left to the
applicant's discretion although Q1 B specifies that
the light sources should produce combined visible
and ultraviolet (UV,320–400 nm) outputs and that
exposure levels should be justified
 The initial trial should have the aim to come upon

32.  Some scientists have found it practical to begin with
extreme conditions such as 80°C or even higher
temperatures and testing at shorter (2,5,8,24h,etc.) multiple
time points, so that the rate of degradation can be evaluated
(The primary degradants and their secondary degradations products can
be distinguished by testing at early time points and thus help in a better
degradation pathway determination)
 In another approach degradation is started by considering
the drug substance to be liable and doing degradation at
the condition mentioned in previous slide. Then stress would
be increased or decreased to obtain sufficient degradation

33. As compared to harsher conditions and
less time approach, this strategy is better
due to the following reasons
 (a) there may be a change in mechanism of reaction when a
harsh condition is used
 (b) there is a practical problem in neutralizing or diluting
every sample, when it contains a high concentration of
reactants, e.g., acid or base, before an injection can be
made on the HPLC column.
Both these reasons are strong enough to
suggest that as normal as possible conditions
should be used for causing the decomposition
of the drug
Studies should be repeated when formulations
or methods change because the change may

34. Which concentration of drug should be used for degradation
study has not been specified in regulatory guidance.
It is recommended that the studies should be initiated at a
concentration of 1mg/mL
 But why ?
it is usually possible to get even minor decomposition
products in the range of detection
It is suggested that some degradation studies should also be
done at a concentration which the drug is expected to be
present in the final formulations

35. Degradation conditions can be classified as follow

36.  Hydrolysis is one of the most common degradation
chemical reactions over a wide range of pH . Hydrolysis is
a chemical process that includes decomposition of a
chemical compound by reaction with water. Hydrolytic
study under acidic and basic condition involves catalysis
of ionizable functional groups present in the molecule.
 Acid or base stress testing involves forced degradation of
a drug substance by exposure to acidic or basic
conditions which generates primary degradants in
desirable range
 If the compounds for stress testing are poorly soluble in
water, then co-solvents can be used to dissolve them in
HCl or NaOH. The selection of co-solvent is based on the
drug substance structure.
 Stress testing trial is normally started at room temperature
and if there is no degradation, elevated temperature (50–
70°C) is applied. Stress testing should not exceed more
than 7 days. The degraded sample is then neutralized
using suitable acid, base or buffer , to avoid further

37.  Hydrogen peroxide is widely used for
oxidation of drug substances in forced
degradation studies but other oxidizing
agents such as metal ions, oxygen, and
radical initiators (e.g., azobisisobutyroni-
trile, AIBN) can also be used.
 Selection of an oxidizing agent, its
concentration, and conditions depends on
the drug substance. 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

38.  The photostability testing of drug
substances must be evaluated to
demonstrate that a light exposure does not
result in unacceptable change.
Photostability studies are performed to
generate primary degradants of drug
substance by exposure to UV or
fluorescent conditions. Some
recommended conditions for photostability
testing are described in ICH guidelines
 The most commonly accepted wavelength
of light is in the range of 300– 800 nm to
cause the photolytic degradation

39.  Thermal degradation (e.g., dry heat and wet heat)
should be carried out at more strenuous
conditions than recommended ICH Q1A
accelerated testing conditions
 Samples of solid-state drug sub- stances and
drug products should be exposed to dry and wet
heat, while liquid drug products should be
exposed to dry heat
 Studies may be conducted at higher
temperatures for a shorter period
 Effect of temperature on thermal degradation of a
substance is studied through the Arrhenius
equation:
k ¼ Ae
where k is specific reaction rate, A is frequency
factor, Ea is energy of activation, R is gas
constant(1.987cal/ deg mole)and T is absolute
-Ea/rt

40. Document degradants an mechanism (prepare report and share
degradation structures, mechanism, in degradation database)
Identify degradants (utilize LC-MS,LC-NMR, preparative isolation: column
chromatography, TLC perp-LC and synthesis to identify unknown degradants)
Select key degradants/track peaks (determine the key degradants
{primary degradants :10% of total degradants across orthogonal methods)
Evaluate purity/potency (obtain purity/potency data including mass balance
where appropriate. Determine purity of the main band using diode array and LC-MS)
Challenge methodology (perform HPLC screening of the degradation samples
using suitable screening method)
Perform experiments (sample at appropriate point using reasonable stress
condition)
Design protocol (develop forced degradation protocol based on the chemistry (CAMEO) of
the API /drug product formulation)
Predict degradants (predicts most likely degradants using the degradation database,
CAMEO and organic chemistry knowledge)

41.  Forced degradation studies provide knowledge about
possible degradation pathways and degradation products
of the active ingredients and help elucidate the structure of
the degradants.
 Degradation products generated from forced degradation
studies are potential degradation products that may or
may not be formed under relevant storage conditions but
they assist in the developing stability indicating method
 It is better to start degradation studies earlier in the drug
development process to have sufficient time to gain more
information about the stability of the molecule.
 This information will in-turn help improve the formulation
manufacturing process and determine the storage
conditions .
 As no specific set of conditions is applicable to all drug
products and drug substances and the regulatory guidance
does not specify about the conditions to be used, this
study requires the experimenter to use common sense .
 The aim of any strategy used for forced degradation is to
produce the desired amount of degradation i.e.,5–20%.
 A properly designed and executed forced degradation

42.  The purpose of stability testing is 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
 The stability testing program also includes the
study of product-related factors that influence its
quality, for example, interaction of the API with
excipients, container-closure systems and
packaging materials

43.  The selection of potential attributes
and time points to be tested should be
justified.
 The retest period or shelf life
assigned to the API by the API
manufacturer should be derived from
stability testing data.

44.  By performing the stability testing of the
API one can identify the likely
degradation products
 {which in turn help in the establishment
of the degradation pathways and the
intrinsic stability of the molecule and
validate the stability-indicating power of
the analytical procedure used}
 The nature of the stress testing will
depend on the individual API and the
type of FPP involved.

45. For an API the following
approaches may be used:
 when available, it is acceptable to provide the relevant data
published in the scientific literature to support the identified
degradation products and pathways
 when no published data are available, stress testing should be
performed.
Stress testing may be carried out on a single batch of the API
It should include the effect of temperature {50°C-60°C}, humidity {75%
RH or greater} and where appropriate, oxidation and photolysis of the
API.
The testing should also evaluate the susceptibility of the API to
hydrolysis across a justified range of pH values when in solution or
suspension

46.  The objective of stress testing is to identify primary degradation
products and not to completely degrade the API.
 The conditions studied should cause degradation to occur to a
small extent, typically 10–30% loss of API as determined by
assay when compared with non-degraded API
 Results from these studies will form an integral part of the
information provided to regulatory authorities

47.  Three batches are chosen
 The batches should be manufactured at
a minimum of pilot scale by the same
synthesis route as production batches,
and using a method of manufacture and
a procedure that simulates the final
process to be used for production
batches.

48.  The stability studies should be conducted
on the API packaged in a container closure
system that is the same as, or simulates,
the packaging proposed for storage and
distribution.

49.  Stability studies should include testing of
stability-indicating attributes of the API, i.e.
those that are susceptible to change during
storage and are likely to influence quality,
safety and/or efficacy. The testing should
cover, as appropriate, the physical,
chemical, biological and microbiological
attributes

50.  Tablets : Dissolution, disintegration, water content and
hardness/friability. Dispersible tablets should additionally be
tested for disintegration (with a limit of not more than 3
minutes) and fineness of dispersion
 Capsules: (hard gelatin capsules) brittleness, dissolution,
disintegration, water content and level of microbial
contamination
(soft gelatin capsules) dissolution, disintegration, level of
microbial contamination, pH, leakage and pellicle formation.
 Oral solutions, suspensions and emulsions
Formation of precipitate, clarity (for solutions), pH, viscosity,
extractables, level
of microbial contamination

51.  Powders and granules for oral solution or suspension:
Water content and reconstitution time
 Metered-dose inhalers and nasal aerosols: Dose content
uniformity, labeled number of medication actuations per
container meeting dose content uniformity, aerodynamic
particle size distribution, microscopic evaluation, water
content, leak rate, level of microbial contamination, valve
delivery (shot weight), extractables / leachables from
plastic and elastomeric components, weight loss, pump
delivery, foreign particulate matter and extractables/
leachables from plastic and elastomeric components of
the container, closure and pump. Samples should be
stored in upright and inverted/on-the-side orientations

52.  Nasal sprays: solutions and suspensions : Clarity (for solution), level of
microbial contamination, pH, particulate matter, unit spray medication
content uniformity, number of actuations meeting unit spray content
uniformity per container, droplet and/or particle size distribution, weight
loss, pump delivery, microscopic evaluation (for suspensions), foreign
particulate matter and extractables/leachables from plastic and
elastomeric components of the container, closure and pump
 Topical, ophthalmic and otic preparations:
Included in this broad category are ointments, creams, lotions, pastes, gels,
solutions, eye drops and cutaneous sprays
 Topical preparations should be evaluated for clarity, homogeneity, pH,
suspendability (for lotions), consistency, viscosity, particle size
distribution (for suspensions, when feasible), level of microbial
contamination/sterility and weight loss (when appropriate).
 Evaluation of ophthalmic or otic products (e.g. creams, ointments,
solutions and suspensions) should include the following additional
attributes: sterility, particulate matter and extractable volume.
 Evaluation of cutaneous sprays should include: pressure, weight loss,
net weight dispensed, delivery rate, level of microbial contamination,
spray pattern, water content and particle size distribution (for
suspensions

53.  Suppositories: Disintegration and dissolution (at
37 °C) and as appropriate for the type, net filled
content, rupture time, melting and solidification,
liquefaction/softening time, leakage, pellicles and
pH.
 Small volume parenterals (SVPs) Color, clarity
(for solutions), particulate matter, pH, sterility,
endotoxins.
 Large volume parenterals (LVPs)
Color, clarity, particulate matter, pH, sterility,
pyrogen/endotoxin and volume.
 Transdermal patches: In vitro release rates,
leakage, level of microbial contamination/sterility,
peel and adhesive forces.

54.  For long-term studies, the frequency of
testing should be sufficient to establish the
stability profile of the API
 For APIs with a proposed retest period or
shelf life of at least 12 months, the
frequency of testing at the long-term
storage condition should normally be every
three months over the first year, every six
months over the second year, and annually
thereafter throughout the proposed retest
period or shelf life.

55.  In general, an API should be evaluated under storage conditions
(with appropriate tolerances) that test its thermal stability and, if
applicable, its sensitivity to moisture. The storage conditions and
the lengths of studies chosen should be sufficient to cover storage
and shipment
Study Storage condition Minimum time period
covered by data at
submission
Long-term • 25 °C ± 2 °C/60% RH ±
5% RH or
• 30 °C ± 2 °C/65% RH ±
5% RH or
• 30 °C ± 2 °C/75% RH ±
5% RH
12 months or 6 months
Intermediate • 30 °C ± 2 °C/65% RH ±
5% RH
6 months
Accelerated • 40 °C ± 2 °C/75% RH ±
5% RH
6 months

56.  Active pharmaceutical ingredients intended for storage in a freezer
 Drug substances intended for storage in a refrigerator
Study Storage conditions Minimum time period
covered by data at
submission
Long term −20 °C ± 5 °C 12 months or 6 months
Study Storage conditions Minimum time period
covered by data at
submission
Long term 5°C ± 3°C 12 months
Accelerated 25°C ± 2°C/60% RH ± 5%
RH
6 months

57.  When the available long-term stability data on primary
batches do not cover the proposed retest period or shelf
life granted at the time of approval, a commitment should
be made to continue the stability studies post-approval in
order to firmly establish the retest period or shelf life.
Commitment that can be made
 if the submission includes data from stability studies on
three production batches, a commitment should be made
to continue these studies through the proposed retest
period or shelf life
 if the submission includes data from stability studies on
fewer than three production batches, a commitment
should be made to continue these studies through the
proposed retest period and to place additional production
batches, up to a total of at least three, in long-term
stability studies through the proposed retest period or
shelf life

58.  if the submission does not include stability data on
production batches, a commitment should be made
to place the first three production batches on long-
term stability studies through the proposed retest
period or shelf life.
 The stability protocol used for long-term studies for
the stability commitment should be the same as that
for the primary batches, unless otherwise
scientifically justified.

59.  A storage statement should be
established for display on the label
based on the stability evaluation of the
API. Where applicable, specific
instructions should be provided,
particularly for APIs that cannot tolerate
freezing or excursions in temperature.
Terms such as “ambient conditions” or
“room temperature” should be avoided.

60.  The purpose of the ongoing stability Program is
to monitor the API and to determine that the API
remains, and can be expected to remain, within
specifications under the storage conditions
indicated on the label, within the retest period or
shelf life in all future batches. The ongoing stability
program should be described in a written protocol
and the results presented in a formal report that
should be available on site.

61.  The purpose of a stability study is to establish data , based
on testing a minimum of three batches of the drug substance
or product, a retest period or shelf life and label storage
instructions applicable to all future batches manufactured
and packaged under similar circumstances.
 The degree of variability of individual batches affects the
confidence that a future production batch will remain within
acceptance criteria throughout its retest period or shelf life.
 Decision Tree for Data Evaluation for Retest Period or
Shelf Life Estimation for Drug Substances or Products
(excluding Frozen Products)