STABILITY STUDY OF VACCINES

DR. M. Masoom Akhtar

Stability of vaccines: key definitions

Stability is the ability of a vaccine to retain its chemical, physical, microbiological and biological
properties within specified limits throughout its shelf-life.

Real-time/ real condition stability studies:

Physio-chemical, biological & other vaccine characteristics during & up to the expected
shelf-life and storage periods under expected handling and storage conditions.

What is a Stable Vaccine?

A vaccine with a very long shelf life:
A vaccine lot that complies with release specifications throughout shelf life.
Technical specifications:
Vaccine that complies with the principles of the initial license throughout life cycle
technical, non-clinical and clinical specifications.

Factors Having a Strong Impact on

Stability of Vaccines
Purity
Formulation
Stabilizer
Human Serum Albumin (HAS), recombinant human Albumin (rHA), Gelatine, Sugars,
Sorbitol, AlPO Ions, Thiomersal,
AlOH (Buffer systems).

Pharmaceutical form
Lyophylized versus liquid
Storage
Frozen versus refrigerator

Guidelines on Stability of Vaccines

ICH HARMONISED TRIPARTITE GUIDELINE QUALITY OF BIOTECHNOLOGICAL
PRODUCTS:
STABILITY TESTING OF BIOTECHNOLOGICAL/BIOLOGICALPRODUCTS, Q5C, dated
30 November 1995.
Development of a CPMP Points to Consider on Stability and Traceability Requirements for
Vaccine Intermediates, dated 2000.
WHO guideline on stability evaluation of vaccines, dated 2006.
Other regional Guidance provide general instructions.
Product specific cases must be considered case by case.
(CPMP-Committee for Proprietary Medicinal Products)
ICH - The International Council for Harmonization of Technical Requirements for
Pharmaceuticals for Human Use (ICH) is an initiative that brings together regulatory authorities
and pharmaceutical industry to discuss scientific and technical aspects of pharmaceutical product
development and registration.

Product Specific Issues

To what extend may critical quality attributes change during shelf life without affecting safety
and efficacy?
Are all potential changes detectable by technical means?
How many stability lots are needed to address the impact on stability due to variances inherent to
the manufacturing process.
Wide specifications
Number and age of intermediates
How can these uncertainties best be investigated during clinical development and post-marketing
monitoring

Stability evaluation: guiding principles

Vital part of quality and safety assessment of a vaccine
Temperature sensitivity
Specific biological activity: potency assay
Design of stability studies - depends of the objectives of stability studies:
Determine shelf-life, storage conditions and to support licensing
Monitor vaccine stability in the post-licensure period.
Support manufacturing changes: comparability

Defining acceptance criteria for stability parameters by clinical trials

The vaccine life cycle

Registration/launch is the critical time:
Studies prior to that point are aimed at obtaining information to support registration.
Subsequent studies are aimed at assuring that the assessments at the time of registration are still
correct

Using stability data and specifications to set shelf life

Goal: Throughout its shelf life, product must be comparable to batches shown to be safe and
effective in clinical studies
Stability data are used to make predictions that can be extrapolated to future batches of product.
The most accurate predictions are based on mathematical modeling of biologically relevant
stability-indicating parameters

Potency of vaccine - Definition

Specific ability or capacity of the product, as indicated by appropriate laboratory tests or by
adequately controlled clinical data obtained through the administration of the product in the
manner intended, to effect a given result

Why do we do a potency assay?

In development, to:
Assure that safe potencies are not exceeded in clinical trials obtain information that will support
licensure-including correlation of potency with clinical response
After licensure, to assure that lots behave similarly to those tested in the clinical trials that
supported licensure.
The potency should not be below the lowest potency believed to be efficacious and the potency
should not exceed the highest potency believed to be safe.
The potency assay thus provides a “bridge” between licensed material and the clinical trials.

What does a potency assay tell us

The assay estimates the mean potency value for a lot.
There are two sources of variability in the measured potency of an individual vial from the same
vaccine lot– manufacturing variability and assay uncertainty.
Thus, we can never know the actual potency in an individual vial that was used in a clinical trial.
We can know the characteristics of the lot of vaccine from which that vial came (we routinely
estimate the mean potency).

Variability in potency assay results

The standard error of the mean potency of a lot provides information about the accuracy with
which we can estimate the mean potency, and thus provides information about both of these
sources of variability, combined
The SEM provides information about our level of certainty that we know the mean potency of
the lot; just as the mean potency does not provide information about individual vials (which is
impossible to obtain, when assays are variable), the SEM provides information about that
estimate
The standard deviation of the mean potency provides information about the distribution of
potency test values, but does not tell us about the underlying actual values.
Conformance to GMP and assuring consistency of manufacture provides assurance that the
manufacturing variability remains constant over time

Clinically relevant parameters

L.L.: the lowest dose at which we are comfortable, based on the clinical data, that the vaccine is
going to be effective. This is normally a specification.

U.L.: The highest dose at which we are comfortable, based on the clinical data, that the vaccine
is going to be safe. This is normally a specification.
Dose
Stability profile
Analytical assay variability
Confidence limits

Two models
Compliance model – Substantially all potency measurements should exceed some predefined
quantity.
 Often, the mean potency from clinical trials is used to set this minimum specification
This is very difficult (perhaps impossible) to reliably implements in original clinical tests were
performed based on an understanding of mean potency, not on an understanding of “all potency
measurements.”
Estimation model – The mean potency of substantially all vaccine lots should be at a level that is
not less than the mean potency of vaccine lots shown to be effective in clinical trials.

Compliance model
Shelf life is set arbitrarily depending on the luck associated with each individual assay at each
time point, and based on the starting potencies of lots on test.
Release potency must be high enough to prevent test results that fall below L.L. due to assay
variability, from requiring an unacceptably short shelf life, or from subsequently failing stability
monitoring studies.
Estimation method
Statistical methods (when appropriate, a regression line analysis) also can be used to estimate
lower bounds on the mean potency estimate at end-expiry for any given release potency, thus
allowing estimation of shelf life. Modeling can be performed after three or more stability time
points have been obtained.
Development-what do we need to know about stability?
What are the kinetics of decay?
What are the degradation products?
Are there stability-influencing factors that should also be controlled?
Do we have sufficient knowledge of the stability of intermediates?
Are the assays adequate?
Are potency assays stability-indicating?
Are potency assays precise enough to support product development?
Do we have a sufficient understanding of material tested in clinical trials?
Do we have a sufficient understanding of product performance at potency ranges likely to be
encountered post-licensure?

Types of stability study

• Real time / real condition stability studies
• Accelerated (degradation) studies
• Stress Testing
• Thermal stability testing
• Supporting stability study
• Stability studies for utilization period
• Impact of container and closure system
• Stability of a vaccine in the case of known “short time excursions” outside the labeled
storage conditions
• Stability study to support selection of VVM

TYPES OF STABILITY
Real Time Stability: Long term stability under recommended storage conditions for the shelf life
proposed for the product.
Accelerated Testing: Studies under exaggerated storage conditions.
Stress studies: More severe conditions than those used for accelerated studies.
Real time / real condition stability studies
Studies on the physical, chemical, biological, biopharmaceutical and microbiological
characteristics of a vaccine, during and up to the expected shelf-life and storage periods of
samples under expected handling and storage conditions.

Accelerated stability studies

Studies designed to determine the rate of change of vaccine properties over time as a
consequence of the expose to temperatures higher than those recommended for storage. Those
studies may provide useful support data for establishing the shelf- life or release specifications
but should not be used to forecast real time real condition stability of a vaccine. They could also
provide preliminary information on vaccine stability at early developmental stages and assist in
assessing stability profile of a vaccine after manufacturing changes.

Stress testing
Studies performed to determine the impact of extreme environmental factors such as light and
extreme temperature (may include oxidizing agents, freeze thaw, pH). These studies are not
usually performed as part of a stability program, but are used instead to establish protective
packaging and container conditions, and to support exclusionary labeling.

TEMPERATURES
Real Time: Storage Temperature of the product e.g. 2-8°C for Tetanus Vaccine
Accelerated: 15±2°C higher than the maximum temperature of storage at real time storage of the
product e.g. 25 ± 2°C
Stress: +10°C ± 2°C than the accelerated temperature e.g.35 ± 2°C
Real Time: 2-8°C for 4 years (for 3 years expiry)
0, 3M, 6M, 9M, 12M, 18M, 24M, 36M & 48M
Accelerated Temperature: 20-25°C for 6 months
0, 1M, 2M, 3M & 6 Months
Stress Study: 35°C for 1-2 months
0, 1, 2, 3, 4, 5, 6 ,7 & 8 weeks
SAMPLE
DRUG SUBSTANCES:
INTERMEDIATE PRODUCT: ACTIVE RAW MATERIAL
FORMULATED BULK: FINAL BULK

DRUG PRODUCT:
FINISHED PRODUCT: FINAL LOT
DESIGN
Bracketing: Extremes of certain design factors, e.g.
Strength: 10 ug, 25ug, 100 ug
Package size: 1 dose, 5 dose, 10 dose
Matrixing: A selected subset of the total number of the possible samples for all factors is tested
at specified time and at a subsequent time another subset of samples for all factors is tested.
Stability indicating parameters
Choice of parameters to be tested:
Depends of the;
Product characteristics
Link between vaccine quality and efficacy or safety
as demonstrated in clinical trials – ideal case.
Potency, antigen content, appearance, pH, completeness of adsorption, adjuvant content;
physicochemical, properties, etc.
New vaccines: parameters to be identified during the product development

Real Time: 2-8°C for 4 years (for 3 years expiry)

0, 3M, 6M, 9M, 12M, 18M, 24M, 36M & 48M
Accelerated Temperature: 20-25°C for 6 months
0, 1M, 2M, 3M & 6 Months
Stress Study: 35°C for 1-2 months
0, 1, 2, 3, 4, 5, 6, 7 & 8 weeks

ACCEPTANCE CRITERIA
In general, significant change is defined as:
5% change in assay from the initial value or failure to meet the acceptance criteria by biological
or immunological parameters or as applicable for the specification of the product.
Any degradation product (impurity) exceeding its acceptance criteria

ACCEPTANCE CRITERIA
Failure to meet the acceptance criteria for physical and chemical parameters
Failure to meet the acceptance criteria for dissolution for stipulated dosages.
Cumulative age of an antigen in the final product
Reported as a major issue in practice
Final product should be stable during the whole period of shelf-life irrespective of the age of its
intermediate products
Data should be collected on an ongoing basis – usually not available before approval of storage
periods and shelf-life
Accelerated stability studies - to demonstrate that an aged intermediate did not affect stability of
the final product
Thermal stability testing – lot release
An indicator of consistency of production of the real time.
TS is a shelf-life specification in current WHO vaccine specific recommendations
Live attenuated vaccines – OPV, MMR, YF
For other vaccines: consider relevance of the rate of change for safety and efficacy
Thermal stability testing
Stability of a vaccine after exposure to a temperature, higher than those recommended for
storage, for a specified period of time, often expressed in terms of change in potency.
Example:
OPV: potency shall not be reduced more than 0.5 log CCID50

Thermal stability testing for lot release

Thermal stability should be considered as a vaccine characteristic that provides an indicator of
consistency of production in the context of lot release.
Thermal stability test is not designed to provide a predictive value of real time stability but to test
a conformation with defined specification for a tested vaccine.

Stability studies for utilization period

Stability studies done to determine time period during which a liquid or a reconstituted
preparation of the final vaccine in an opened container can be used.
Example: after reconstitution and kept at room temp/2-8O C

Impact of container and closure system. For liquid form products

This should be tested by exposing and maintaining samples into different positions during a
certain period of time. (Upright, horizontal or inverted position).
These positions should mimic possible situations that may occur during the transport and storage
and that provide contact between vaccine and the closure system

Stability of a vaccine in the case of known ‘’short time excursions’’ outside the labeled storage
conditions
Possibility of temperature excursion during transportation
Take the samples out of cold chain (2-8°C) and put them at 25°C for a short period such as 1 ay
or 8 hours and then return them back to the normal cols chain condition.
Samples can be taken out of the cold chain at the first period and/or the middle period and/or the
last period of shelf life after released
The release specification may be calculated by adding the correction factor from this stability
study.
Stability study to support selection of VVM
A vaccine vial monitor, or VVM, is a circular indicator, printed directly on the vaccine vial label
or affixed to the top of the vial or ampoule. The inner square of the VVM is made of heat-
sensitive material that is initially light in color and becomes darker when exposed to heat over
time.

Stability study to support selection of VVM

Stability study is to determine the change rate of potency that is related to change of VVM
indicator color at different temperatures and time exposure.
The rate of potency change which related to VVM color change is a product specific
characteristic.
Four categories of VVMs are needed because of the different temperature and time sensitivities
of EPI vaccines including
– VVM2 for the least stable vaccines (2 days to end point at +37OC)
– VVM7 for moderate stability vaccines (7 days to end point at +37OC)
– VVM14 for medium stability vaccines (14 days to end point at +37OC)
– VVM30 for high stability vaccines (30 days to end point at +37OC)
Stability study protocol
Minimum three lots.
Where the same strength and container is used for three or more fill contents so the smallest and
the largest container size are considered as representative of all.
Pilot scale data may be acceptable providing that manufacturing scale batches are tested
following approval and comparability demonstrated.

Stability study protocol

Stability indicating parameters are defined on case by case basis, mostly include potency/ antigen
content, appearance, pH, general safety, specific toxicity, antimicrobial agent content,
completeness of adsorption, sterility, adjuvant (adsorbent) content, and changes in physico-
chemical properties.
Quantitative assay is required to detect the rate of change

Stability study protocol

Testing frequency (3, 6, 9, 12, 18 and every 6 months afterwards) described pharmaceuticals
does not apply to all vaccines. The appropriate time points testing should be set up based on
characteristics of the vaccine, the rate of change of the parameter measured, the purpose of
testing, study design and subsequent data analysis.

Stability of Intermediates & Cumulative Age

The stability of intermediate is required if the intermediate is not used in the next step of
production immediately.
Total age of all components at the end of shelf-life is considered as cumulative age.
Stability data of the final product should include the data generated on the intermediates of
different ages used in the final formulation.

Stability of combined vaccines

Determination of the shelf-life should be based on the shortest shelf-life component
Data obtained for monovalent vaccines should support stability of combined vaccine
Stability of combined vaccines should not be based on the extrapolation of the stability data of
the individual components alone
Labelling
Recommended storage conditions and expiry date
– Sensitivity of vaccines to environmental factors such as light, freezing and preventive
measures
If VVMs are to be used, adequate stability data should be generated to support selection of
appropriate VVM for the vaccine in question