Stability kinetics and testing

Presented by
(Dr) Kahnu Charan Panigrahi
Asst. Professor, Research Scholar,
Roland Institute of Pharmaceutical Sciences,
(Affiliated to BPUT)
Web of Science Researcher ID: AAK-3095-2020
12/14/2021 KC PANIGRAHI 1

KINETICS OF STABILITY

• Kinetics deals with the study of the rate at which processes occur
and mechanism of chemical reactions.
• It is determined by the change in the concentration of the
reactants or products as a function of time.
• The rate may be determined by the slowest or rate determining
step.
• The number of concentrations that determine rate is the order of
reaction.
• The number of concentrations term that bring about the reaction is
called molecularity of reaction.
dt
dc
 Rate  kc
n
Stability Kinetics

ZERO ORDER REACTION:
Rate is constant and is independent of the concentration of any of the reactants. A
constant rate of drug release from a dosage form is highly desirable.
Reactant [A] Product(P)
Rate = - dc/dt = K [c]0
- dc/dt = k
dc = - k dt
t
ct
dc   kdt
c0 t0
co = Initial concentration
ct = Concentration at time
Ct –C0 = -kt

c = co –Kt
K = (co –c) /t
K = Concentration / time
= mole/liter.second
= M. sec-1
5
C
t
Units of the rate constant K
12/14/2021 KC PANIGRAHI
5

Determination of t1/2
Let c = co /2 and t1/2 = t substitute in
equation; c = co –kt
t1/2 = co / 2K
Note: Rate constant (k) and t1/2 depend on co
Determination of t0.9
Let c = 0.9 co and t= t0.9
substitute in equation; c = co – k t
t90% = t0.9 = 0.1 co /k
6

- dc/dt = kc = kc
- dc/c = kdt
c t
 c
 k dt
dc
c0 t0
ln c ln co  kt
0
kt
2.303
log c  log c 
FIRST ORDER REACTION:
The reaction rate of change is proportional to drug concentration.

C = co e –kt
Difficult to determine
slope
lnc = lnco – kt
Slope = c1 – c2 / t1 – t2
Slope = - k
lnco
log co
log c = log co – kt / 2.303
Slope = c1 – c2 / t1 – t2
Slope = - k / 2.303
C lnc
logc
t t
t
12/14/2021
KC PANIGRAHI

Determination of t1/2
Let t = t1/2 and C = C0 /2
substitute in equation ln C = ln C0 –Kt
t1/2 = ln 2/ K
= 0.693 / K
K units = time-1
Determination of t0.9
Let t = t0.9,c = 0.9co
substitute in equation
ln c = ln co –Kt
t0.9 = 0.105 / K
t1/2 = 0.693 / K
t0.9 = 0.105 / K

Accelerated Stability Testing

OBJECTIVE OF STABILITY TESTING:
• “……To provide evidence on how the quality of a drug
substance or drug product varies with time under the
influence of a variety of environmental factors such as
temperature, humidity & light, & enables recommended
storage conditions, re-test periods & shelf lives to be
established”
(ICH) 2003

 Stability of a pharmaceutical preparation is the capability of a formulation in a
specific container-closure system to remain within its physical, chemical,
microbiological, therapeutic and toxicological specifications throughout its shelf life.
 Stability testing is used to:
• Provide evidence as to how the quality of the drug product varies with time.
• Establish shelf life for the drug product.
• Determine recommended storage conditions.
• Determine container closure system suitability.
 Accelerated stability study to predict the shelf life of the product, by accelerating the
rate of decomposition, preferably by increasing the temperature of reaction
conditions.
 Preparations are subjected to high stresses during stability testing. Common high
stresses include : Temperature, Humidity, Light
INTRODUCTION

Study Storage condition Minimum time period
covered by data at
submission
Long Term
(Ambient)
25º C ± 2º C
60%RH ± 5% 12 months
Intermediate
(controlled)
30º C ± 2º C
60%RH ± 5%
6 months
Accelerated 40º C ± 2º C
75%RH ± 5%
6 months
Testing Frequency:
•For Long term testing, during first year sampling should be done every three
months, during second year, sampling should be done every six months and after
two years, sampling should be done once a year.
•Accelerated testing should be done at least six months and it suggests sampling
points of 0, 3, 6 months.

Arrhenius equation
Arrhenius factor
Energy of activation
It explains the effect of temperature on rate of a reaction. According to
Arrhenius, for every 10º rise in temperature, the speed of reaction
increases about 2-3 times.
k = A e -Ea /RT
Ideal gas constant
Arrhenius factor is the frequency of molecular collisions occuring between the
molecules.
Activation energy (Ea) is the minimum energy that a molecule should possess so that
the molecular collisions produce the product.

Estimation of k value
• The Preparation is stored at different elevated temperatures, to accelerate the
degradation.
• Samples are withdrawn at different time intervals
• Concentration of reactants is determined
• Appropriate graphs are drawn for the kinetic data.
• The Order of the reaction is determined by plotting the appropriate function of
concentration against time and linear relationship is determined.
• Straight line in a graph permits the estimation of k value from the slope
Similarly graphs are drawn for different elevated temperatures.
0
kt
2.303
log c  log c 

Estimation of Self life
• By using Arrhenius relationship, Log k values are plotted against reciprocal of absolute
temperature (1/T) .
• A straight line is obtained, the slope of the line is negative and the magnitude is Ea / 2.303
R.
• The intercept corresponds to log A.
• Extrapolate the straight line to room temperature (k25) and read the log k value on y-axis.
• Substitute the k value in the appropriate equation to get the shelf life of the product.
log k = log A – Ea / 2.303 RT

Arrhenius plot for predicting the rate constant at ambient
temperature(25ºC).

Limitations
• Stability predictions based on Arrhenius equation are valid only when the break down
depends on temperature.
• The energy of activation obtained in the study should be between 10 to 30 kcal/mole.
• It cann’t be applied:
 when degradation is due to microbial contamination photochemical reactions
 When the product looses its physical integrity at higher temperatures.
 When the order changes at elevated temperatures.
 In case of disperse systems, when temperature is elevated viscosity is decreases and this
may introduce errors in the prediction of stability.

SUBJECTS FOR DISCUSSION
KINETICS OF STABILITY
OBJECTIVE
ABBREVIATIONS
ESSENTIAL ICH DEFINITIONS
ACCELERATED STABILITY TESTING
STABILITY TESTING OF APIS
STABILITY TESTING OF FPPS
BRACKETING AND MATRIXING
PHOTOSTABILITY TESTING
CONCLUSION
REFERENCE DOCUMENTS

ABBREVIATIONS
DRA Drug Regulatory Authority
ICH International Conference on Harmonization
GMP Good Manufacturing Practices
API Active Pharmaceutical Ingredient
FPP Finished Pharmaceutical Product

SELECTED DEFINITIONS
 Shelf life (also referred to as expiration dating period):
The time period during which a drug product is expected to remain within
the approved shelf life specification, provided that it is stored under the
conditions defined on the container label.
 Re-test date:
The date after which samples of the drug substance should be examined to
ensure that the material is still in compliance with the specification and
thus suitable for use in the manufacture of a given drug product.
 Re-test period:
The period of time during which the drug substance is expected to remain
within its specification and, therefore, can be used in the manufacture of a
given drug product, provided that the drug substance has been stored
under the defined conditions.

• Primary batch:
A batch of a drug substance or drug product used in a formal stability
study, from which stability data are submitted in a registration
application for the purpose of establishing a re-test period or shelf life.
• Pilot scale batch:
A batch of a drug substance or drug product manufactured by a
procedure fully representative of production scale batch.
• Production batch:
A batch of a drug substance or drug product manufactured at production
scale by using production equipment in a production facility as specified
in the application

ZONE TEMPERATURE YEARLY AVERAGE
HUMIDITY (%RH)
Zone I ( Moderate) 21 ̊C 45
Zone II
(Mediterranean)
25 ̊C 60
Zone III (Hot, dry) 30 ̊C 35
Zone IV (Very hot,
moist)
30̊ C 70
Worldwide zones and the temperature
and humidity conditions

REGIONS ZONE I &II ZONE III&IV
EUROPE All countries
AMERICA Argentina, Bolivia, Canada,
Mexico, US
Brazil, Columbia, Cuba,
Jamaica
ASIA Afghanistan, China, Iran,
Nepal, Turkey
INDIA, Bahrain , Hong
Kong, Oman , Pakistan,
Srilanka, UAE
AFRICA Egypt, Algeria, South
Africa, Libya
Angola, Benin, Congo,
Uganda, Sudan, Somalia,
Senegal
Countries belonging to various zones

 General
 Stress Testing
 Selection of Batches
 Testing Frequency
 Evaluation
STABILITY TESTING OF APIS

General:
 For active substances not described in an official pharmacopoeial
monograph, stability studies are required.
 For active substances described in an official pharmacopoeial monograph,
for which suitable limits have been set but a re-test period is not defined,
two options are acceptable:
 The manufacturer of the pharmaceutical product confirms that the active
substance complies with the pharmacopoeial monograph immediately prior
to the manufacture of the pharmaceutical product. In this case no stability
studies on the active substance are required.
 The manufacturer establishes a re-test period based on the results of long
term testing stability studies conducted on the active substance.

Stress Testing:
For an API the following approaches may be used:
• When available, it is acceptable to provide relevant data published in the
scientific literature to support the identified degradation pathways and products.
• When no data is available, stress testing should be performed.
.

The Role of Stress Testing:
Stress testing of the active substance can help in
• Identification of degradants
• Identification of degradation pathways
• Determination of which type(s) of stress affect the molecule:
– Photostability
– High Temperature
– Low Temperature
– Oxidation
– pH extremes
– Water

Typical Stress conditions:
Stress factor Conditions
Heat 10°C increments
Humidity 75%RH or greater
Acid 0.1N Hcl
Base 0.1N NaOH
Oxidative 3%H2O2
Photolytic Xenon,Metal hailde lamp or Near
UV,White florescent lamp

Selection of Batches:
• Data from formal stability studies should be provided on at least three
primary batches of the active substance.
• The batches should be manufactured to a minimum of pilot scale by the
same synthetic route as, and using a method of manufacture and
procedure that simulates the final process to be used for, production
batches.

Specification:
• Stability studies should include testing of those attributes of the drug
substance 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.
e.g. appearance, assay, degradation.

Testing Frequency:
For long term studies:
Year 1: every 3 months
Subsequent years: annually
At accelerated storage conditions: (6 month study)
Minimum three points including t0 and tfinal,
e.g. 0 3 6
(initial) (final)
At intermediate storage conditions : (12 month study)
Four points including t0 and tfinal,
e.g. 0 6 9 12
(initial) (final)

• If long-term studies are conducted at 25°C ± 2°C/60% RH ± 5% RH and
“significant change” occurs at any time during six months’ testing at the
accelerated storage condition, additional testing at the intermediate
storage condition should be conducted and evaluated against significant
change criteria.
significant change – failure to meet its specification
Study Storage condition
Minimum time period
covered by data at
submission
Long Term*
(Ambient)
25º C ± 2º C
Intermediate**
(controlled)
30º C ± 2º C
65%RH ± 5%
6 months
75%RH ± 5%
6 months
General case:

Drug substances intended for storage in a refrigerator:
Minimum time period
covered by data at
submission
Long Term 5º C ± 3º C 12 months
60%RH ± 5%
6 months
• If significant change occurs between three and six months’ testing at the
accelerated storage condition, the proposed re-test period should be
based on the real time data available at the long term storage condition.

• In the absence of an accelerated storage condition for active substances
intended to be stored in a freezer, testing on a single batch at an
elevated temperature (e.g. 5°C ± 3°C or 25°C ± 2°C or 30°C ± 2°C) for an
appropriate time period should be conducted to address the effect of
short term excursions outside the proposed label storage condition.
e.g., during shipping or handling
Minimum time period
covered by data at
submission
Long Term -20º C ± 5º C 12 months
Drug substances intended for storage in a freezer:

Evaluation:
• Minimum of 3 batches of drug product was tested
• The analyst must found the batch to batch variability & if it is small than
only it is accepted & it can be done by different statistical test's ( P value
for level of significance for rejection).
• Where the data show so little degradation and so little variability then it
is normally unnecessary to go through the formal statistical analysis;
providing a justification for the omission should be sufficient.

 In general, “significant change” for a drug product is defined as:
1. A 5% change in assay from its initial value;
2. Any degradation product’s exceeding its acceptance criterion;
3. Failure to meet the acceptance criteria for appearance, physical attributes,
and functionality test (e.g., color, phase separation, resuspendibility, caking,
hardness, dose delivery per actuation); however, some changes in physical
attributes (e.g., softening of suppositories, melting of creams) may be
expected under accelerated conditions;
and, as appropriate for the dosage form:
4. Failure to meet the acceptance criterion for pH; or
5. Failure to meet the acceptance criteria for dissolution for 12 dosage units.

STABILITY TESTING OF FPPS
 General
 Selection of Batches
 Testing Frequency
 Evaluation

General:
 The design of the formal stability studies for the pharmaceutical product
should be based on knowledge of -the behaviour and properties of the
active substance, -from stability studies on the active substance,
-on experience gained from pre-formulation studies and
investigational pharmaceutical products.

Selection of Batches:
• Data from stability studies should be provided on at least three primary
batches of the pharmaceutical product.
• The primary batches should be of the same formulation and packaged in
the same container closure system as proposed for marketing.
• Two of the three batches should be at least pilot scale batches and the
third one can be smaller, if justified.
• Where possible, batches of the pharmaceutical product should be
manufactured by using different batches of the active substance.

Testing Frequency:
For long term studies:
Subsequent years: annually
At accelerated storage conditions: (6 month study)
Minimum three points including t0 and tfinal,
e.g. 0 3 6
(initial) (final)
At intermediate storage conditions : (12 month study)
Four points including t0 and tfinal,
e.g. 0 6 9 12
(initial) (final)

• If long-term studies are conducted at 25°C ± 2°C/60% RH ± 5% RH and
“significant change” occurs at any time during 6 months’ testing at the
accelerated storage condition, additional testing at the intermediate
storage condition should be conducted and evaluated against significant
change criteria
Minimum time period
covered by data at
submission
Long Term*
(Ambient)
25º C ± 2º C
Intermediate**
(controlled)
30º C ± 2º C
65%RH ± 5%
6 months
75%RH ± 5%
6 months
General case:

• A 5% loss in water from its initial value is considered a significant change
for a product packaged in a semi-permeable container after an
equivalent of 3 months’ storage at 40°C/NMT 25% RH.
Minium time period
covered by data at
submission
Long Term*
(Ambient)
25º C ± 2º C
Intermediate**
(controlled)
30º C ± 2º C
65%RH ± 5%
6 months
NMT25%RH
6 months

• If significant change occurs between 3 and 6 months’ testing at the
accelerated storage condition, the proposed shelf life should be based on
the real time data available from the long term storage condition.
Minimum time period
covered by data at
submission
Long Term 5º C ± 3º C 12 months
60%RH ± 5%
6 months
Drug products intended for storage in a refrigerator:

• For drug products intended for storage in a freezer, the shelf life should
be based on the real time data obtained at the long term storage
condition.
Drug products intended for storage in a freezer:
Minimum time period
covered by data at
submission
Long Term -20º C ± 5º C 12 months

Evaluation:
• Minimum of 3 batches of drug product was tested
• The analyst must found the batch to batch variability & if it is small than
only it is accepted & it can be done by different statistical test's ( P value
for level of significance for rejection).
• Where the data show so little degradation and so little variability then it
is normally unnecessary to go through the formal statistical analysis

 In general, “significant change” for a drug product is defined as:
1. A 5% change in assay from its initial value;
2. Any degradation product’s exceeding its acceptance criterion;
3. Failure to meet the acceptance criteria for appearance, physical attributes,
and functionality test (e.g., color, phase separation, resuspendibility, caking,
hardness, dose delivery per actuation); however, some changes in physical
attributes (e.g., softening of suppositories, melting of creams) may be
expected under accelerated conditions;
and, as appropriate for the dosage form:
4. Failure to meet the acceptance criterion for pH; or
5. Failure to meet the acceptance criteria for dissolution for 12 dosage units.

Stability-indicating quality parameters
Stability studies should include testing of those attributes of the FPP that are
susceptible to change during storage and are likely to influence quality, safety
and/or efficacy.
For instance, in case of tablets:
♦ appearance ♦ hardness
♦ friability ♦ moisture content
♦ dissolution time ♦ degradants
♦ assay ♦ microbial purity

Dosage form Evaluation
Tablets
Appearance,colour,odour,assay, degradation
products, dissolution,moisture and friability.
Hard gelatin capsules
Appearance,colour,odour,assay,degradation
products,dissolution,moisture and microbial
limits
Soft gelatin capsules
Appearance,colour,odour,assay,degradation
products,dissolution,moisture and microbial
limits,pH,leakage.
Emulsions
Appearance,colour,odour,assay,
degradation products, microbial
limits,PH,viscosity,preservative content and
distribution of dispersed phase globules.

Oral solutions
Appearance,colour,odour,assay,degradatio
n products, PH,microbial limits,
preservative content.
Oral suspensions
n products, PH,microbial limits,
preservative
content,redispersibility,rheological
properties, mean size and distribution of
particle.
Oral powders Appearance,colour,moisture,and
reconstitution time.
Inhalations and nasal sprays
n products, dose content uniformity,
microscopic evalution,water content, leak
rate, microbial limits.

Topical,opthalamic,ointments
,creams,
lotions,pastes,gels,solutions.
Appearance,clarity,colour,
homgeneity,odour,pH,resuspendibility,visco
sity,particle size distribution, assay,
degradation products,preservatives,
microbial limits, weight loss
Small volume parenterals
Appearance,colour,clarity,
assay,presarvative content, degradation
products, particulate matter, sterility,
Large volume parenterals
Appearance,colour,clarity,
assay,presarvative content,degradation
products,particulate
matter,sterility,pH,pyrogenicity, volume.

ACCELERATED STABILITY
 This stability study run under more stressful conditions than expected for
long term storage to account for any changes outside the label storage
conditions
e.g. during shipping or handling.
 Acc Storage condition should be guided by intended climatic condition in
which API/FPP will be stored.
 The goal is to get a quick understanding of what may be expected from a
long term study.
Long-term conditions Accelerated Conditions
Room temperature (25-30°C) 40°C ± 2°C/75% RH ± 5%
Refrigerated (5°  3°C) 25°C ± 2°C/60% RH ± 5% RH or
30°C ± 2°C/65% RH ± 5%
Freezer (-20°C 5°C) Can range from 5°C ± 3°C or 25°C
± 2°C or 30°C ± 2°C

PHOTOSTABILITY TESTING
Photostability testing studies include:
• Test on drug substance.
• Test on exposed drug product outside the immediate pack.
• Test on drug product in the immediate pack.
• Test on drug product in the marketing pack.
Light source
Option 1: Artificial daylight lamp combining both visible & UV output similar to
D65 & ID65.
Option 2: Cool white fluorescent & near UV lamp(320-400nm)
output similar to that
specified in ISO 10977
max. energy emission
between 350-370 nm

Procedure:
determine the absorbances of the sample (AT) and the control (Ao) at
400 nm using a 1 centimeter (cm) path length
Eg: Quinine chemical actinometry:
2%w/v aq.solution of quinine
monohydrochloride dihydrate
10ml of sol. in a 20 ml colorless
ampoule wraped in aluminum foil
(CONTROL)
10ml of sol. in a 20 ml colorless
ampoule
(SAMPLE)
Expose to the light source
Change in absorbance calculated by
Δ A = AT - Ao.

 To Evaluate the photosensitivity
 In elucidating degradation pathway
 In validating analytical procedures
 Information necessary for
handling, packaging and labeling
 To study, identify precautionary
measures needed in manufacturing
or in formulation
PHOTOSTABILITY
TESTING
Forced
degradation
studies
Confirmatory
studies

BRACKETING
• It is the design of a stability schedule such that only samples on the
extremes of certain design factors, e.g., strength, package size, are tested
at all time points as in a full design.
• The design assumes that the stability of any intermediate levels is
represented by the stability of the extremes tested. Where a range of
strengths is to be tested, bracketing is applicable if the strengths are
identical or very closely related in composition.
e.g: for a tablet range made with different compression weights of a
similar basic granulation.

BRACKETING DESIGN
Pack type
Label strength and batch
numbers (X,Y,Z)
10 mg 20mg 30mg
X Y Z X Y Z X Y Z
Alu/Alu blister cards of
10 tablets
+ + + - - - + + +
HDPE pack of 30
tablets
+ + + - - - + + +
HDPE pack of 100
tablets
- - - - - - - - -
HDPE pack of 1000
tablets
+ + + - - - + + +
HDPE- high density polyethylene + indicates- Sample tested

MATRIXING
• It is the design of a stability schedule such that a selected subset of the
total number of possible samples for all factor combinations is tested at a
specified time point.
• At a subsequent time point, another subset of samples for all factor
combinations is tested.
• The design assumes that the stability of each subset of samples tested
represents the stability of all samples at a given time point.

MATRIXING
Example of matrixing design on time points for a product in two
strengths (S1 and S2) are shown in Table:
• The term “one-half reduction” refer to the reduction strategy initially
applied to the full study design.
• A “one-half reduction” initially eliminates one in every two time points
from the full study design.
• The reductions are less than one-half due to the inclusion of full testing
of all factor combinations at some time points.
• These example include full testing at the initial, final, and 12-month time
points.
• The ultimate reduction is therefore less than one-half (24/48) and is
actually 15/48.

MATRIXING DESIGN
one-half reduction
Time points
(months)
0 3 6 9 12 18 24 36
S
1
Batch 1 + + - - + - + +
Batch 2 + - + + + - + +
Batch 3 + - + - + + - +
S
2
Batch 1 + - + + + + - +
Batch 2 + + - - + + - +
Batch 3 + + - + + - + +
+ indicates- Sample tested
Example of Matrixing Design on Time Points for a Product with
Two Strengths

CONCLUSION
• Stability studies should be planned on the basis of
pharmaceutical R&D and regulatory requirements.
• Forced degradation studies reveal the intrinsic chemical
properties of the API, while formal stability studies establish
the retest date.
• The shelf life (expiry date) of FPPs is derived from formal
stability studies.

REFERENCES
1) ICH Q1A (R2): Stability testing of new drug substances and products
(http://www.ich.org/LOB/media/ MEDIA419.pdf).
2) ICH Q1B: Photostability testing of new drug substances and products
(http://www.ich.org/LOB/media/ MEDIA412.pdf).
3) ICH Q1C: Stability testing of new dosage forms
(http://www.ich.org/LOB/media/MEDIA413.pdf).
4) ICH Q1D: Bracketing and matrixing designs for stability testing of new drug
substances and products(http://www.ich.org/LOB/media/MEDIA414.pdf).
5) ICH Q1E: Evaluation for stability data
(http://www.ich.org/LOB/media/MEDIA415.pdf).
THE FOLLOWING ICH GUIDELINES MAY BE CONSULTED IN THE CONTEXT OF
STABILITY TESTING:

REFERENCES
6) The WHO stability guideline –
Stability testing of active pharmaceutical ingredients and finished
pharmaceutical products
(WHO Technical Report Series, No. 953, Annex 2, 2009)
7) V.SAI KISHORE’s Drug regulatory affairs
(pg no 227-288)

THANK YOU

Stability kinetics and testing

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