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sas-Farmer, senior director of bioanalytical labs with Frontage Laboratories.
Furthermore, analytical method development for APIs tends to be more
straightforward because reference materials with known concentrations are
available and can be used to spike into
the matrix. With biomarker assays, internal standards are not often available,
and, generally, an endogenous level of the
biomarker is already in the matrix, which
makes quantitation a challenge, according to April Brys, director of biomarker
services with Battelle.
Regulatory requirements for analytical methods also differ for APIs and
biomarkers. API analytical method development
and validation must meet US
Cynthia A. Challener, PhD
Pharmacopeial Convention requirements
or guidelines set by the International
Conference on Harmonization (ICH).
Biomarker assay development and
validation, meanwhile, depends on the
assay’s intended use. If it is being used
as a diagnostic tool, it must meet requirements of the Clinical Laboratory
Improvement Amendments (CLIA). If
it is being used to establish a pharmacokinetic/pharmacodynamics (PK/PD)
endpoint, it must meet good laboratory
practice (GLP) standards.
Many of the same analytical techniques, however, are used for API
methods and biomarker assays. “Smallmolecule biomarker assays are typically
developed using a liquid chromatography/mass spectrometry (LC/MS) platform, which provides a great balance of
sensitivity, selectivity, robustness, precipproaches to drug development are challenging for biomarkers than it is for sion, accuracy, and ease of operation,”
changing, as pharmaceutical com- APIs. This article will examine some of states Patrick Bennett, executive director
panies use advanced technologies these issues.
of biomarker operations at PPD. The LC/
to achieve targeted goals. Today, ratioMany of the challenges of working MS platform is also beneficial for bional drug development strategies based with biomarkers stem from the fact that, marker analysis because it can be used
on modeling, simulation, and biomark- unlike APIs in neat solutions, they are en- to perform multi-component analyses
ers complement such traditional drug dogenous compounds in biological fluids. simply, including metabolomics-based
discovery methods as structure-activity For example, isobaric analytes such as analyses of hundreds of molecules per
relationship (SAR) studies. Biomarkers bile acids in matrices can lead to higher sample.
are also being used to evaluate drug background noise and interference in
performance during preclinical testing biomarker assays. In addition, biomark- Dealing with matrix issues
and clinical trials. Assay development ers are typically present in trace amounts, For biomarker assays, regulatory agenand validation, however, can be more unlike APIs, according to Stephanie Pa- cies prefer that calibration standards and
Cover Story: Biomarker Assays
Developing and Validating
Assays for Small-Molecule
Biomarkers
A
SCIENCE PHOTO LIBRARY - TEK IMAGE/GETTY IMAGES
Working with biological matrices and
understanding the intended use are crucial.
Cover Story: Biomarker Assays
quality control samples be prepared using
the same biological matrix (whether
blood, urine, plasma, or other biological fluid) found in the samples to be
analyzed. However, they should be free
of biomarker, which can influence the
accuracy of readings. Unfortunately, it is
difficult to obtain analyte-free matrices.
One option, according to PasasFarmer, is to use a surrogate matrix that
is biomarker free. A second option is
to remove the endogenous analyte/biomarker from the matrix through chemi-
cal entity is in the early stages of development. Bennett notes, for example, that,
even if no compliance is deemed necessary, extensive method performance
qualification may still be warranted to
ensure that the biomarker assay can
provide adequate confidence in the data
required.
Validation of an API analytical method
is typically focused on the validation of
Intended use and validation
specific assay performance parameters
Before method validation, it is crucial to such as the precision, robustness, linidentify the intended use of the biomarker earity, and lower limit of quantification
(LLOQ)/lower limit of detection (LLOD).
In contrast, validation of biomarker assays involves determination of “fitness for
purpose” and must demonstrate that the
assay meets the predetermined purpose,
according to Brys, who defines the stages
of biomarker validation as follows:
• Defining the purpose and selection
of the assay
cal treatment, charcoal stripping, selec- assay. “The investigator must understand
• Identifying the critical reagents and
tive extraction, or some other method. the ultimate use of the data generated,”
standards
In both of these cases, however, the says Pasas-Farmer. If biomarker data are
• Writing the procedure and validachosen matrix will be different from the to be used for early screening and evalution plan
study sample matrix. “With surrogate, ation of the mechanism of action (MOA)
• Performing the actual study validastripped, and depleted matrices, there of a drug candidate, then a qualified assay
tion, which typically includes asmay be issues with solubility, stabil- that is semi-quantitative or relatively
sessment of fitness for purpose, robustness of the assay in the clinical
ity, non-specific binding, or ionization quantitative can be used, she says, but if
setting, standardization of patient
matrix effects due to the differences in the biomarker data are used to support
the modified matrix and the authentic
matrix,” says Bennett. Consequently, it
will be necessary to design validation experiments to confirm that the ability to
quantify analytes in a modified matrix is
similar to that for analytes in the authentic matrix. If an authentic matrix is combined with standard addition, Bennett
adds, experiments and processes will be
necessary to determine the baseline and
sampling, and the collection, storage,
final concentrations and generate bridg- claims for the safety/efficacy of dosing
in a pivotal preclinical or clinical study,
and stability of the samples.
ing data from newer lots.
• Evaluating the assay during routine
These issues may be avoided by using and if they will be used in a regulatory
use
a standard addition approach with the filing in support of a new drug applica• Quality control monitoring
true biological matrix once the basal tion (NDA), then a fully validated assay
• Proficiency testing
levels of the endogenous biomarker have should be implemented.
• Identification of batch-to-batch isFinally, if the assay will be used as a
been calculated for the specific lot of
sues.
control matrix. Stable, labeled reference diagnostic tool to determine the disease
If stable labeled material is used as a
material can be used as a surrogate stan- state of an individual, then CLIA validadard, and labeled internal standards can tion requirements must be met, she ex- surrogate reference standard, Bennett
says, additional experiments must be perbe effective. These approaches, however, plains.
Discussions are still ongoing, however, formed immediately before each analytican also be challenging because, often,
reference standards are not available, or about whether biomarker assays should cal run, to demonstrate similar responses
are difficult to synthesize, for biomarker be fully validated, or if qualification of an for equimolar amounts of labeled and unassays. Even if they are available, whether assay can still be used if the new chemi- labeled analyte. Because biomarkers are
they are labeled or unlabeled, they can
be expensive.
In some cases, depending on the detection method and the type of matrix,
samples may have to be purified before a
biomarker assay can be completed. This,
in turn, can create various problems, including recovery and matrix interference
issues, notes Brys.
Many of the challenges of working with
biomarkers stem from the fact that, unlike
APIs in neat solutions, they are endogenous
compounds in biological fluids.
FDA’s recent guidance suggests that the
agency will take a more rigorous and less
semi-quantitative approach to overseeing
assay qualification and validation.
Cover Story: Biomarker Assays
endogenous and the goal is to measure
the change in the biomarker concentration in response to some other change
(e.g., use in diseased vs. healthy patients
or in comparing different therapies), he
says, experiments must also be run to determine the ability to detect a statistically
meaningful change.
must be in place for initial discovery into
preclinical, followed by preclinical into
clinical, as each stage will have its own
level of rigor, and follow the fit-for-purpose assay qualification and validation
steps,” Eash notes.
Given the low levels of biomarkers
present in most samples, Brys adds, it
is important to identify an analytical
Successful strategies
method with sufficient sensitivity. Access
While it might seem obvious, Bennett to actual study or patient samples can be
stresses that, even before assay develop- important during the pre-qualification/
ment starts, it is crucial to ensure that the validation stage to ensure that the right
Regulatory expectations for biomarker
methods are changing, and are expected
to continue to evolve over the next three
to five years.
-Patrick Bennett, PPD
correct biomarker(s) is being evaluated
for the right endpoint(s). Brys adds that
selected biomarkers should have a strong
biological rationale and correlation with
efficacy in preclinical studies and, thus,
focusing on a few biomarkers is more
effective. “A basic starting point for the
development of any biomarker assay is an
understanding of relevant precedents for
the assay,” agrees Donna Eash, director of
client services with Frontage.
As important is ensuring that the
extent of qualification or validation is
aligned with the objective of the biomarker data that is obtained, according
to Bennett. “If the data are used for dose
selection, formulation comparisons, or
research, then compliance may not be as
important as ensuring that the method
meets an appropriate acceptance criteria. However, if the data are to be used
for patient enrollment, safety, or efficacy,
meeting required regulatory compliance
standards also should be included in the
strategy,” he observes.
Developers must also outline an effective development plan, considering that
endogenous biomarkers are often identified in discovery stages. “A good strategy
concentration range(s) are being evaluated and to determine differences in the
concentrations of the biomarker(s) in
treated and untreated samples, according
to Bennett. He also reiterates that validation experiments should be performed to
quantify any matrix effects and non-specific binding that may occur when using
modified or surrogate matrices.
The following are also important to
the successful development and validation of biomarker assays, Brys says:
• Evaluating biomarker stability, particularly during storage
• Proper determination and evaluation of sample collection and storage methods
• For biomarker assays that are intended for use in diseased patients,
the validation process must also
include screening patients to evaluate how the biomarker is affected by
the disease state and how the disease
state will affect the analysis, according to Pasas-Farmer.
Multiplexing and other advances
New advances in analytical technology,
such as multiplexing systems that allow
for 10–100 biomarkers to be analyzed in
a single sample, offer a number of benefits, including lower costs and fewer
invasive procedures, says Pasas-Farmer.
High resolution/accurate mass (HR/
AM) instruments are now commercially
available with triple quadrupole-like sensitivity and robustness. Bennett says that
they offer significantly higher resolving
power with a much greater signal-tonoise ratio, even when using single-ion
monitoring (SIM). SIM is advantageous
because it does not require the fragmentation and subsequent loss of signal that
occurs in LC-MS/MS experiments. This
means that, for many analytes, particularly those with poor or excessive fragmentation or high background noise
levels, even when MS/MS is used, it
allows for greater selectivity and sensitivity, he says. Examples would include
bile, which has poor fragmentation, and
steroids, with excessive fragmentation.
Easier and more robust nanospray
technologies have also allowed researchers to achieve significantly better LLOQs
than was possible with high-flow liquid
chromatography and traditional electrospray ionization techniques, Bennett
says. Pairing this technology with either
HR/AM or triple quadrupole instruments can provide sensitivity benefits
that are further magnified when multidimensional chromatography and an
immunoaffinity cleanup step are used,
he adds.
Evolving regulatory environment
Regulatory expectations for biomarker
methods are changing, and Bennett expects them to continue to evolve over
the next three to five years. FDA’s recent guidance suggests that the agency
will take a more rigorous and less semiquantitative approach to overseeing
assay qualification and validation. In
addition, Incurred Sample Reanalysis,
which is used to measure the quality
of data and robustness of an assay and
has been required for small- and largemolecule drug substances, is now being
requested for biomarker development,
Pasas Farmer points out. PT
Posted with permission from the February 2015 issue of Pharmaceutical Technology ® www.pharmtech.com. Copyright 2015, Advanstar Communications, Inc. All rights reserved.
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