Drug Discovery & Development
Clinical
stress test
Increasing demands on the pharmaceutical industry to develop
drug candidates for clinical therapy quickly have challenged
scientists to expedite the drug discovery and development
process. Robyn A Rourick, James Gallagher and John P
Walsh, Kalypsys Inc Pharmaceutical Sciences, discuss
the integration of a modular chemical and photo-stability
chamber for profiling degradants in drug substances.
Conditions used for
compound degradation:
■ Acidic ■ Peroxide
■ Basic ■ Light
M
any new paradigms for accelerating drug development
focus on increased productivity using benchmarks
such as the number of preclinical lead candidates,
faster IND/NDA filings and efficient technology transfer.
Clearly, these initiatives have had a profound effect on
the analytical laboratory requiring
significant increases in samples,
broader diversity of compounds and
shorter analysis timelines.
How analytical groups effectively and
efficiently absorb these changes while
maintaining avenues for diversity and
scientific growth is a significant challenge.
These changes, distinct and measurable,
are likely to be continual and will also
result in improved analytical approaches
and schemes for analysis. Resources
have been leveraged to exploit the area
of automation towards project-oriented
objectives strategically aimed at accelerating
drug development.
Understanding compounds
An early understanding of the chemical
and photo stability of a drug substance
plays a central role in these project-
oriented activities. Understanding a
compound’s sensitivity to light or
chemical stability is an important
consideration from a practical and a
regulatory perspective in
pharmaceutical development. Poor
photo or chemical stability of a
WPF014_040_LookingatLab.indd 57
compound may trigger additional regulatory concerns about
toxicity of observed degradants. As lead compounds are
progressed into a regulated manufacturing environment,
analytical separations must evolve to accurately track the
process and ensure a deep understanding of process impurities
and degradation products. In addition to reactants and
products, a strong drug substance method will monitor the
degradation profile of the target compound.
The generally accepted technique for determining
the required resolution of a separation
involves subjecting the target compound
to aggressive environments to force the
degradation of the compound. These
conditions are typically: acidic, basic,
peroxide and light.
Author profiles
James Gallagher is associate director, pharmaceutical sciences,
Kalypsys Inc. His responsibilities involve analytical and
pharmaceutical development activities in support of early phase
clinical programmes. Gallagher has held positions at Pfizer, La Jolla
Laboratories, Amylin Pharmaceuticals and Carnation Company.
Robyn A Rourick is director, pharmaceutical sciences, Kalypsys
Inc. Prior to Kalypsys, Rourick was associate director of DuPont
Pharmaceuticals Research Labs and group leader, analytical
technologies at CombiChem Inc. She has also held positions at
Bristol-Myers Squibb Pharmaceutical Research Institute.
John P Walsh joined Kalypsys Inc in 2003. He is responsible
for developing and implementing novel analytical and
bioanalytical technologies, which impact or enhance drug
discovery efforts. Currently, Walsh is leading several research
initiatives to support IND filings.
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WPF014_040_LookingatLab.indd 58
The process of generating degraded samples, reducing data
and collating results is traditionally time- and labour-intensive.
Historically, every stability experiment has been treated as
a one-off by preparing each manually in a scintillation vial
requiring large amounts of sample. In order to understand
compound liabilities in-house, prior to contracting the work,
a cost-effective, high(er) throughput method of conducting
these stability tests was initiated. This new approach to
understanding degradation is project based and benefits
from workflow integration, project management tools for
information management and data warehousing.
The system
Using CAD software, an in silica model was designed in-house
and optimised to achieve industry standards; including the
ICH Q1A and Q1B Guidances for Industry on Stability and
Photostability testing. Criteria for the chamber required a
unique design; liquid handler accessibility, meeting industry
device qualification standards, temperature control of the
samples, active ventilation and division of stability chambers to
provide independent sample degradation.
A dual chamber design for the stability chamber was produced
to separate the workflow to prevent cross-contamination and
allow for concurrent use of the chamber. The chamber was
designed to reside on the Tecan Genesis deck, with robotic arm
access to each of the assay compartments (Figure 1, page 60).
This robotic access and integration with the liquid handler
capabilities of the system provided effective sample
management during a run of the assay.
Industry typically uses pre-certified stability chambers that
resemble upright refrigerators, and can cost up to $180,000.
Other custom stability solutions have yielded robotic systems
equivalent to ultra high throughput screening systems in every
form: size, price and complexity. The stability testing targeted
was segregated into two types: chemical and photo. Each
would be pursued as a separate means of analysis; individual
spatial regions and unique sample handling. An additional
feature of the Tecan deck was the fact that the selected
platform allows for automated sample analysis by direct
injection of degraded samples into a MUX LCT Premier.
The photostability chamber was qualified to ICH Q1B Option
1 standards, via recommended lamp choice and the quinine
actinometric method. The chemical stability chamber enforces
elevated sample temperature via a Peltier thermal device, which
the sample block rests on. The sample block uses glass vials to
eliminate vessel leeching under the harsh chemical conditions;
20 vials allow for four compounds under five conditions. The
photo stability chamber provides for appropriate sample photo
degradation in 72 hours with only 40mg of sample consumed.
With the chemical stability chamber’s elevated sample
temperature and highly caustic conditions, appropriate
degradation occurs within four hours.
The size of the stability chamber allowed for all components
of the system to remain on the deck and maintain the status
12/9/08 15:57:59

of the unit as a modular addition (Figure 2, page 60). This
direction for design was taken to allow for other assays such
as solubility to continue their analysis on the deck, without
being hindered by the stability chamber’s integration.
The ability of the Tecan to access the chamber, and by
consequence the samples, allows for on-line LC/MS analysis.
The samples can be injected into the LC/MS directly from
the liquid handler. The key benefits of this online LC/MS
analysis are: quench-free stability analysis and exact time
points for kinetic measurements. In most degradation
experiments, the solutions must be neutralised prior
to analysis to prevent further degradation, as the samples
must wait until they are analysed. In this case, the samples
can be injected directly onto the LC/MS in a matter of
seconds, reducing out-of-experiment degradation and
highly accurate time points.
Application of photostability assessment
It is the responsibility of the sponsor to ensure that the
formulation and packaging can adequately protect a
photolabile product from light during manufacturing,
packaging and storage. A means to perform a rapid
assessment of a compound’s photostability can provide
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Drug Discovery & Development
A means to perform a rapid
assessment of a compound’s
photostability can provide an
early alert to liabilities.
an early alert to the liabilities and guide a sponsor to
undertake appropriate protections for its compound
throughout its shelflife. For example, tetracycline requires
light protection measures such as amber lighting in
manufacturing suites, packaging in amber glass and
restrictions on sunlight exposure on ingestion.
Drug product formulations should also be assessed
for photostability during development to assess any
photostability concerns. Designing a formulation that
may inhibit the photodegradation of an active compound
is also a consideration by incorporating approaches such
as encapsulation, TiO2 coating or the use of a vehicle that
inhibits absorption of light energy or prevents degradation.
Once a photostability concern has been identified, having
a means to determine the rate of degradation is important
in finalising the formulation and package as well as setting
operational parameters during processing.

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12/9/08 15:58:20
 Drug Discovery & Development
Figure 1. A robotic arm enters the chamber with samples.
The procedure for rapid assessment of stability is
summarised as follows:
1. Drug substance was evaluated using automated photo
and chemical stability chamber.
2. Degradants were characterised in a rapid, accurate
manner using LC/MS approaches.
3. Drug product formulation was evaluated as well
as potential storage conditions.
4. Supportive data was used in IND filing, to support
formulation claims and labelling for clinical trials.
Developments ahead
Stress testing continues to be a key component of the
drug development process. It serves as the main tool
to understand and predict stability problems as well
as to aid in the development of analytical methods and
to identify degradants and pathways.
Efforts by the International Conference on Harmonization
regarding impurities and photostability have raised
regulatory scrutiny of impurities, requiring elucidation and
toxicological qualification at very low levels. Currently there is
no regulatory guidance mandating how stress testing should
be performed and, as a result, approaches are tailored by the
expertise of the company or scientists directing the studies.
At Kalypsys, a custom chemical and photo-stability chamber
has been developed and integrated with a Tecan liquid
handler with rapid LC/MS capabilities in order to produce a
streamlined approach for identifying degradative liabilities in
pharmaceutical compounds. This automated platform allows
for the exposure of compounds in 96-well plates to acid,
base, light and temperature conditions, and allows for the
rapid generation of samples in support of stability indicating
method development and validation. The degradation
methods are simple to implement and modify, allowing a
wide variety of conditions to be examined in a timely manner.
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WPF014_040_LookingatLab.indd 60
Figure 2. The size of the system allows all components to remain on the deck.
Additional advances involve data analysis and interpretation
of the separation data. Analysis tools are still in the early
stages but promise great utility. Storage of conditions,
retention times and other useful data in a database allow
for data-driven decisions around separation enhancement.
The data also provide a backdrop of information relevant
during method transfers to CMO/CROs or during
process improvements, which reduce method development,
and qualification hurdles. wpf
FDA guidance for stress testing in chemical and
photo stability chambers.
Chemical stability chamber
FDA Guidance Q1A(R2) Section 2.1.2 states that stress testing should include:
■ the effect of temperatures in 10C increments (for example, 50C, 60C)
above that for accelerated testing
■ the effects of oxidation
■ evaluating the susceptibility of the drug substance to hydrolysis across a
wide range of pH values when in solution or suspension.
Examining degradation products under stress conditions is useful in
establishing degradation pathways and developing and validating suitable
analytical procedures. Results from these studies will form an integral part of
the information provided to regulatory authorities [FDA Q1A (R2) 2.1.2].
Photo stability chamber
FDA Guidance Q1B outlines the appropriate conditions to be met for
appropriate photo stability analysis:
Section I.B Option B details the lamp specification that should be followed;
D65/ID65 emission standard pursuant to ISO 10977 (1993).
■ Iwasaki Metal Halide MTD150/D.
■ Section IV Option 1 was used to validate the chambers performance
using the quinine monohydrochloride dihydrate actinometric
standard specification.
■ Sections II and III are followed for testing drug substance and drug
product, respectively.
The test article must receive a measured dose of both UVA (200 watt-hours per
square metre) and Visible (1.2 million lux-hours) optical radiation exposure.
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