GS Tiến Sĩ
trường Đại Học Dược Khoa Sài
for Drug Stability Testing
Dong To, D.Sc.
In the last step
of the product/process development, the main objectives of
the Formulation group are optimizing the drug delivery
system and increasing the stability of a drug product. When
the process is validated, then the testing of drug stability
in the Manufacturing group will insure that the medicine
retain its identity, strength and quality in the market
throughout the period up to the expiration date.
The knowledge in many areas of
the drug such as potency, metabolism, and
physical/chemical/biochemical pathways allows the rational
development of analytical methods for the expiration dating.
In this overview, some
principles, practices and stability testing guidelines from
the FDA of the US are briefly presented.
I. DRUG STABILITY
It is note that one could find in literature various
examples of drug formulation using trehalose and hydrophobic
sugar glasses (1), liposomes (2, 12), cyclodextrins (3, 19),
of chemical kinetics and drug stability (4, 5, 6, 7), and of
expiration dating and shelf life estimation (8, 9, 10).
Also, one can get considerable amounts of updated
information in the web sites of FDA including the FDA
modernization act of 1997 (FDAMA) (13, 14, 15) and in that
of the European Agency for the Evaluation of Medicinal
products EMEA (16). In addition, one can have relevant
services as provided by many independent labs (17, 18, 19,
The stability means compliance of the drug product within
the specifications. The stability testing insures the
quality of the drug product as defined by its content of
active ingredient, its purity and its organoleptic,
physicochemical and biological properties. The drug could be
a chemical, a biologic or a medical device. In general,
biologics depend mostly on optimum formulation and good
storage conditions for stability.
The methods used to assess and control stability are based
on rate constants of degradation reactions of the drug.
The shelf life of the drug can be calculated if the rate of
loss "k" of the drug with time at storage temperature is
If [A]0 and [A]t are the initial
active concentration and the residual active concentration
at time t, the following rate equations describe the usual
zero order: [A]t
= [A]0 - kt
first order: Ln [A]t
= Ln [A]0 - kt
second order: 1/[A]t =
1/[A]0 + kt
The drug product expiration dates are usually based on
assumed zero- or first-order kinetics. The shelf-life t0.90
is the time at which decomposition reaches 10% or activity
decreases to 90%. The time should be determined at which the
95% one-side lower confidence limit for the mean degradation
curve intersects the lower acceptable specification limit
(appendix 1). This will assure that the average drug
characteristics of the batch are within specifications up to
the end of the expiration period.
II. STABILITY PROTOCOL
1. General Product Information:
The basic information of the drug must be presented:
Container-closure: composition, type and size.
2. Specifications and Test Methodology Information:
The specifications on physical, chemical, biological and
microbiological characteristics of the drug must be
described. A definition of potency is usually needed for
biological activity of a drug.
The analytical methodology should be validated and presented
with method accuracy, precision and suitability (11).
The method measuring the trace amount of harmful by-product
or unwanted degraded product during drug storage should be
known with acceptable limit of detection.
3. Study Design and Study Conditions:
The sampling plan, number of units and sampling times are
selected according to statistical quality control methods.
The testing of drug products for reconstitution at the time
of dispensing (as directed on the labeling) must be defined.
The same requirement is needed after they are reconstituted.
The duration of the study and storage conditions:
temperature, humidity and light should be specified. For
example, the Human Medicines Evaluation Unit of the EMEA
defined significant change as failure to meet the
specifications with long term testing, at temperature 25o
± 2o C and relative humidity 60% ± 5% RH for 12
months, and with accelerated testing at 40o ± 2o
C and 75% ± 5% RH for 6 months.
4. Stability Data/Information:
The lot number from research, pilot or production must be
provided with the corresponding manufacturing date. The age
of the bulk/active substance used in the testing should be
mentioned. The analytical data and source of data points
must be defined.
All relevant information of previous formulations or
container-closure systems should be provided.
5. Data Analysis and Conclusions:
The appropriate statistical methods and formulae used in the
analysis must be documented. The calculations, statistical
analysis and graphs to evaluate data should be provided. The
results of statistical tests for potency estimates as well
as the proposed expiration date and its justification must
The release specifications are defined to warrant an
acceptable minimum potency at the initial release for full
expiration dating period.
III. STABILITY STUDY
1. Real-time stability:
This stability study at storage condition is the most
reliable, but unfortunately it takes a long time and is very
costly in the development phase of the drug.
2. Accelerated stability:
The accelerated stability
study predicts the expiry date using exaggerated storage
conditions. The drug substance can be stressed as many ways
as possible, using temperature, humidity, light, pH,
solvents, buffers etc…
The rate constant k is
observed to have an exponential dependence on temperature.
Where k is the reaction rate constant of any order, A and Ea
are constants, and T is the absolute temperature, according
k = A exp(-Ea/RT)
The activation energy -Ea
can be calculated as equal to R*Slope with R = Gas Constant
= 1.987 cal K-1 mol-1.
The activation energy Ea
is an energy barrier of the system that the reactants must
pass before becoming products. The usual range of Ea
is about 12 to 24 kcal/mol, with hydrolysis: 14-20 kcal/mol
and oxidation: 23 kcal/mol.
The Ea depends on
formulation, for example: the phenylbutazone in water has
three rates: k1, k2, k3 of
oxidation/hydrolysis corresponding respectively to Ea1,
Ea2, Ea3 of 24.4, 26.7, 36.2 kcal/mol.
In solvent such as dimethyl formamide, diethyl carbinol,
propylene glycol, the Ea ‘s are lower, from 3.9
to 7.7 kcal/mol, giving a shorter shelf life, only from 18
days to 113 days.
The QdT factor,
ratio of reaction rates at two temperatures differing by dT
degrees, can be calculated as:
C, knowing shelf life at
higher°This factor is used to predict shelf life at 4 temperature. Usually, Q10
C equals to 2, 3 and 4 with°
to 30°from 20 Ea ‘s from 12.2 to 24.5 kcal/mol. This means that the
rate increases C.°C
from initial rate at 0°6
to 32 times at 2525°C
from initial rate at 0° C.
The rate and
order of reactions of some drugs are presented with kinetic
= [A]o - kt
suspension ® Salicylic acid +Acetic acid
k = 0.0075
mol l-1d-1 or 1.5% d-1
= 6.67 days
Ln [A]t = Ln [A]o - kt
®4-Aminobenzoic acid+ Ethanol
k = 0.05 w-1
= 0.693/k = 13.86 weeks
1/[A]t = 1/[A]o+ kt
Hydroxybenzoic acid diethylamide + Propanol
k = 0.012
mmol l-1 w-1
= 0.926 weeks
order reversible kinetics
Photochemical Isomerisation [A Û B]
Competitive first order degradation
Hydrolysis/Rearrangement [C ¬ A ® B]
Sequential first order kinetics
Rearrangement/Hydrolysis [A ¬ B ® C]
Analysis of a Monoclonal Antibody MoAb degradation study:
of accelerated testing using temperature to predict
stability of a monoclonal antibody MoAb against carcino-embryonic
antigen CEA is described. Samples are stressed at 37oC
and the percent immunoreactivity % IR are recorded as
140mM Phosphate, pH 8.5, 37oC
models are tested and results are tabulated in the following
model describing the degradation of this monoclonal antibody
is the zero order kinetics. It gives the highest correlation
coefficient R of 0.9907989, the closest estimated initial %IR
of 95.9 and the smallest sum of squares SS of 34.1.
immunoreactivity of this monoclonal antibody replaces its
potency in the calculation of loss of activity with time.
The antibody vials at up or inverted positions of storage
are used to detect effect of stoppers, but no difference has
been found. The testing temperatures are 22oC, 37oC
and 45oC. All physical, chemical, biological and
microbiological characteristics of this antibody are tested
at time intervals, but only results for activation energy,
rate constants and shelf-lives at various temperatures
calculated using Excel program are presented in appendix 2.
Some calculation programs used in the Stability Study:
Any of the
following software could be used: RS/1 from BBN, Statistical
Analysis System SAS, Excel PC, Q-basic or Irwin's computer
aspects and examples of the drug stability testing are
presented. The accelerated testing to predict expiry dating
of a monoclonal antibody against CEA is described. In a
dosage form, each drug substance represents a particular
case and should be treated accordingly. A good formulation
could only be obtained by a team effort of many scientists
in the product/process development phase.
1. Trehalose and novel hydrophobic sugar glasses in drug
stabilization and delivery
Gribbon et al., 1996
2. Stealth liposomes, Microencapsulation
3. Cyclodextrins: Drug solubilization and stabilization
Loftsson T. and Brewster M.E., 1996
4. Chemical kinetics and drug stability
Guillory J.K. and Poust R.I., 1996
5. Expiration dating of pharmaceutical compounds in
relation to analytical variation, degradation rate, and
Natarajan J., Altan S. and Raghavarao D., 1997
6. Shelf life estimation for multifactor stability studies
Ahn H. and Tsong Y., 1997
7. Stability Testing of Drug Products
Wissenchaftliche Verlagsgesellschaft mbH Stuttgart 1987
Scientific Criteria, guidelines and official state
requirements in Europe, Japan and USA.
8. Accelerated Testing
John Wiley & Sons, 1990
Statistical Models, Test Plans, and Data Analyses
9. Drug Stability, Principles and Practices
Jens T. Carstensen
Dekker, Inc., 1995
Principles and practices of achieving drug stability
10. Stability of Protein Pharmaceuticals
Center for Professional Advancement
Course Director: Dr. Christopher Rhodes
Drug product stability
Accelerated stability tests.
11. Statistical Methods in Analytical Chemistry
Peter C. Meier and Richard E. Zund
John Wiley & Sons, 1993
12. The Pharmaceutical Journal
journal of the Royal Pharmaceutical Society of Great
Vol 269 No 7216, p: 414-417, 21 September 2002
British Pharmaceutical Conference 2002 summary
of the Center for Devices and Radiological Health (CDRH),
regarding pre-market approval application PMA, PMA
supplements, pre-market notifications (510k) for Devices and
post market surveillance (PS) submissions.
of the Center for Drug Evaluation and Research (CDER),
summarizing the design and analysis of the real-time and
accelerated stability studies applicable to chemicals,
biologics and medical devices.
of the Center for Biologics Evaluation and Research (CBER),
listing Guidance/Guidelines/Points to Consider. The
guidances are listed in order by date, with the most recent
guidance added to the top.
of the European Agency for the Evaluation of Medicinal
products (EMEA), describing Stability Testing Guidelines:
Stability Testing of New Drug Substances and Products. The
Human Medicines Evaluation Unit defined significant change
as failure to meet the specification with long term testing
and accelerated testing. The International Conference on
Harmonization (ICH) presented a Harmonized tripartite
guideline for EC, Japan and USA.
of Magellan, listing all comprehensive analytical services
for drug substance testing. This independent lab offers
degradation chemistry and development of methods for drug
of Cabrillo providing excipient compatibility assessments in
Laboratories Cabrillo Facility offers packages to
support pre-formulation and formulation development.
from CyDex, Inc. specializing in the commercialization of
modified cyclodextrins for use in drug development and
from Quintiles’s Pharmaceutical Sciences: Stability Testing,
Design Stability Protocol for investigational new drug IND
and new drug application NDA… This company provides
extensive service for testing stability and storage
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