1461 ISSN 1757-6180 Bioanalysis (2009) 1(8), 14611474 10.4155/BIO.09.

130 2009 Future Science Ltd

Review
Recent drug development is based on the mech-
anism of action of the drug on specic biological
targets and pathways. Biomarkers reective of
these pathways have been linked to physiologi-
cal data to aid drug-development decisions [1].
There are high expectations in the pharmaceu-
tical industry that biomarker applications will
drive faster and more successful drug develop-
ment [24], as exemplied in the multitudes of
biomarker conferences and publications devoted
to this relatively new application of biomarkers.
Biomarkers expressed in disease-specic path-
ways can provide evidence that a drug hits its
target to exert functional changes. Studies of
the target and proximal biomarkers can provide
pharmacodynamic (PD) information for expo-
sure/effect modeling. Data from downstream
distal biomarkers can provide proof-of-biology
of the drugs effect on disease progression [5].
Protein therapeutics via target-mediated
mechanisms have been successfully devel-
oped. The bioana lysis of protein therapeutics
is based on the evolving practices from small
to large molecules: the US FDA issued guid-
ance for bioanalytical method validation to
support pharmacokinetic (PK) studies with the
focus on conventional small-molecule drugs,
mainly by LCMS methods [6]. Additional
White papers on ligand-binding assays (LBAs)
that are widely used to study biotherapeutics
have been published [79]. At the 3rd American
Association of Pharmaceutical Scientists
(AAPS)/FDA Bioanalytical Workshop, the
validation and implementation of bioanalytical
methods for both small- and macro-molecules
were discussed and consensus reports were sub-
sequently published in a themed issue of the
AAPS journal [911].
The terminology of GLP compliance has been
generally used in the pharmaceutical industry
to indicate bioana lysis in support of PK/toxico-
kinetic (TK) studies that are conducted accord-
ing to guidance from the FDA and/or other
regulatory agencies [6]. It is not uncommon to
see analysts working in the PK/TK arena adopt-
ing the same guidance for biomarker method
validation. At the same time, since biomarker
kits approved by the FDA or other regulatory
agencies have been routinely used for disease
diagnosis, clinical chemists also participate in
biomarker ana lysis for drug development and
perform the assays under regulations from
agencies such as the Clinical Lab Improvement
Amendments in the USA. The end-users of the
data also come from two camps: PK/PD scien-
tists who are familiar with PK-type data and
physicians/clinicians who are comfortable with
the routine clinical chemistry output.
Method validation and application of protein
biomarkers: basic similarities and
differences from biotherapeutics
Protein drug development and biomarkers share common bioanalytical technologies that are applied for different
purposes. A ft-for-purpose approach should be used for biomarker assays at various stages of novel biomarker
development and their application to drug development. Biomarker quantifcations can be absolute or relative,
depending upon the characteristics of the standard curve, which include the reference standard, substituted matrix
and parallelism. Appropriate method-validation experiments should be carried out on sample collection, relative
accuracy and precision, range fnding, parallelism, selectivity, specifcity and stability in order to meet the need for
exploratory or advanced application that is specifed for a study. The interaction of a biotherapeutic with the target
ligand or inter-related biomarkers should be taken into consideration for method platform choice and validation.
Direct adoption of commercial diagnostic kits can produce confounding data. Therefore, kit comparison, modifcation
and appropriate validation experiments are often carried out to meet the specifc purpose for drug development.
Multiplex assays and physicochemical methods can complement the single-analyte ligand-binding assay for protein
drugs and biomarkers.
Jean W Lee
Pharmacokinetics and Drug
Metabolism, Amgen Inc.,
One Amgen Center Drive
30E-3-B, Thousand Oaks,
CA 91320, USA
Tel.: +1 805 447 9463
Fax: +1 805 499 9027
E-mail: jwlee@amgen.com
BiotheRapeutics
Therapeutics derived from
biological products or processes
Ligand-Binding assay
Analytical methods that
determine the analyte using the
signal resulting from the binding
reaction of the reagent and
the analyte
For reprint orders, please contact reprints@future-science.com
Demonstration Discovery Characterization Qualification Surrogacy
Studies of cells, animal model
or human with tight patient control
Confirmatory with small
human population at
multiple sites
Multiple sites
Large sample size
Extended populations
Multiple drugs of similar mechanism
Exploratory method validation Advanced method validation (GLP similar)
Biomarker development
Drug development
Nonclinical Lead optimization Pivotal clinical Post-approval Early clinical
Nonregulated Regulated (GLP) PK bioanalysis
Post-approval
surveillance
Safety and
efficacy biomarkers
Patient stratification
Other therapeutic indications
Market differentiation
Safety biomarkers
Efficacy biomarkers
Proof of Biology
Protocol design
PK/PD modeling
Dose selection
Biomarker panel
selection
Target and
candidate selection
Candidate attrition and refinement
A
B
Review | Lee
Bioanalysis (2009) 1(8) 1462 future science group
The inconsistency in adaptations of regula-
tions in either bioanalytical or clinical laborato-
ries and a lack of regulatory guidance contribute
to the confusion regarding biomarker data qual-
ity required for drug development. A position
paper proposed that biomarker assay validation
and implementation should be t-for-purpose
to produce reliable data appropriate for the
application [12]. Biomarker applications are very
different from those of diagnosis, which pro-
hibit the direct adoption of clinical laboratory
practices. The intended use of biomarker data
should be considered in order to determine the
rigor of method validation and implementation
for the specied purpose. Biomarker ana lysis to
support PDs should be similar to that for PK
studies with differences based upon the unique
endogenous nature of the heterogeneous bio-
marker [1215]. This review focuses on the simi-
larities and differences of protein biomarker
assays compared with those from PK bioana lysis
for biotherapeutic development.
Table 1. Comparison of pharmacokinetic and biomarker bioana lysis.
Intended
application
Method types Pre-analytic
sample
collection
Reference
standard
Analytes Calibrator
matrix
Validation
sample and
QC preparation
Accuracy
PK study
PK parameters
of BA and BE
Mostly denitive
quantication
methods
Test with spiked
standard
Well
characterized
and pure
Exogenous
and well
dened
Analyte-free
biological
matrix
Spiked reference
standard into
biological matrix
Absolute
accuracy
Biomarker study
PD: safety and
efcacy
Denitive,
relative,
quasiquantitative
or qualitative
methods
Consider
pathway
conversion and
artifact from
cell activation;
diurnal effect
Many are not
well
characterized or
pure, may not
be the same
as endogenous
Endogenous,
not well
dened
Substituted
matrix
Spiked reference
standard for VS
and QC, pooled
authentic samples
for sample
controls
Mostly
relative
accuracy
BA: Bioavailability; BE: Bioequivalance; LLOQ: Lower limit of quantication; PD: Pharmacodynamic; PK: Pharmacokinetic; QC: Quality control; VS: Validation samples.
Figure 1. (A) Biomarker- and (B) drug-development processes.
PD: Pharmacodynamics; PK: Pharmacokinetics.
pRotein BiomaRkeR
Protein that is objectively
measured and evaluated as an
indicator of normal biologic
processes, pathogenic processes
or pharmacologic response to a
therapeutic intervention
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Intended purposes of biomarker
bioana lysis are different
from biotherapeutics
Development of novel biomarkers follows phases
of discovery, characterization and clinical qual-
ication/validation (FiguRe 1a) analogous to
those of drug development (FiguRe 1B) [5,16,17].
Initially, biomarkers are discovered for explor-
atory studies in cell systems, animal models or
well-controlled human studies. The data provide
characterization of the biomarker in the path-
way for internal decision making. Application to
advanced studies will test the linkage to clinical
outcome using small sets of patient populations
at multiple sites. Some biomarker results may
show negative or uninterpretable linkage. Others
with promising results may advance to clinical
qualication (validation) studies where extensive
data are collected from multiple sites (with large
patient numbers and extended populations) on
multiple drugs and for multiple indications
involving the same pathway. Surrogacy can only
occur after the accumulation of a huge amount
of data before the biomarker can replace the
clinical outcome. The level of rigor of method
validation and documentation increases from
exploratory to advanced use.
The drug-development phases are depicted in
FiguRe 1B. The drug exposure data determined
in animal TK and PK and human PK help to
dene the therapeutic window and decide the
proper dose and dosing frequency. The use of
biomarkers at various phases of drug develop-
ment is depicted in the boxes below the bar.
These include go/no-go decision making on
candidates, PK/PD modeling to decide dose and
frequencies, patient stratication and safety, and
efcacy monitoring [3,4,1319].
The objective of method validation is to
demonstrate that a particular method is reli-
able for the intended application [6]. Thus, a
t-for-purpose approach for method validation
and sample assays is suitable for both drug and
biomarker bioana lysis. During discovery and
lead optimization of drug development, fast
decision making on multiple drug candidates is
supported by methods with or without minimal
prestudy characterization by non-GLP methods.
To support TK and PK studies, GLP methods
with rigorous method validation are usually
required (FiguRe 1B).
The purposes of biomarkers are more diverse
than those of drug development; however,
method-validation approaches can be roughly
categorized into those of exploratory or advanced
application (FiguRe 1a). FiguRe 2 depicts the basic
concept of t-for-purpose biomarker method val-
idation and how the exploratory and advanced
validations are used during biomarker develop-
ment. The rigor of method development, valida-
tion and documentation for advanced applica-
tion is more intense and GLP similar, except for
a few distinguishing features. The basic similari-
ties and major differences are listed in taBLe 1
and further discussed later in this article.
Pre-analytic considerations that
impact biomarker ana lysis
Pre-analytic considerations for biomarkers are
represented in FiguRe 3. It is necessary to select
the potential biomarkers and the correspond-
ing biological matrix, dene the intended pur-
pose and decide the type of method validation
that suits the application. A work plan may
be used to clarify the purpose and lay out the
experiments to be conducted [20].
Table 1. Comparison of pharmacokinetic and biomarker bioana lysis (cont.).
Selectivity Specicity Assay acceptance
criteria
Stability Reproducibility
PK study
Spike recovery test on
~six matrix lots at LLOQ
and one other level
Test against target ligand, in addition to
similar structures; measurement of free
drug preferred over total
4-6-X rule Use QC samples Incurred sample
repeats
Biomarker study
Spike sufcient amount
over basal level, test
more lots from healthy
and disease populations
Test against drug molecule(s), precursor
and downstream molecules;
measurement of total target biomarker
may be the pragmatic option over the
free form
Depends on drug effect,
disease and biological
modulation and method
performance
Use sample
controls and trend
ana lysis for long
term storage
Sample controls
BA: Bioavailability; BE: Bioequivalance; LLOQ: Lower limit of quantication; PD: Pharmacodynamic; PK: Pharmacokinetic; QC: Quality control; VS: Validation samples.
Discovery
Demonstration
Characterization
Qualification
Surrogacy
Novel biomarker
development
Advanced method validation Exploratory method validation
In-study method validation
Prevalidation
Pre-analytical and analytical method
feasibility method optimization
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Bioanalysis (2009) 1(8) 1464 future science group
Stability of the analytes in stock solution and
biological matrix during the processes of sample
collection, storage, shipping, freezing/thawing
and throughout the last assay should be evaluated
for drug compounds and biomarkers [21]. The
sample collection stability for monoclonal anti-
body drugs in serum has been well established;
however, the stability of peptides often depends
upon blood collection, anticoagulants and time
of exposure to high temperatures. Therefore,
plasma or serum sample collection for novel
peptide biotherapeutics and biomarkers should
be investigated for possible stability issues.
Pre-analytic variables have hindered data
utility in proteomic biomarker discovery and
validation [22,23]. Errors from variable specimen
collection can be higher than those arising from
sample ana lysis itself. The conversion of precur-
sors to the biomarker of interest will lead to
overestimation, while degradation will result
in underestimation of the analyte. Inhibitors
of relevant activation or proteolysis should be
included in the collection syringe or added to
the sample promptly. Some biomarkers can only
be quantied in plasma so as to avoid prote-
olysis or platelet activation of the coagulation
pathway during serum collection. Bulk serum
collected into a bag can result in lower recovery
than in serum from venipuncture used in a clin-
ical study for some biomarkers [24]. The shear-
ing effect through a small bore needle or the
use of high-speed centrifugation on blood cells
may cause endothelial cell activation, resulting
in analytical artifacts. For biological uids of
relatively low protein content (e.g., urine and
cerebral spinal uid), collection tubes, transfer
pipettes and storage containers must be evalu-
ated to minimize adsorption of a peptide/
protein to the contact surfaces.
It is important to standardize techniques for
all sample collection and handling and to keep
these consistent throughout the duration of the
use of the assay [25,26]. For example, the G-force
and revolution per minute conversion should
be dened for each laboratorys centrifuge in
order to avoid mistakes. The standard processes
of collections from multiple sites, barcodes and
transports to the analytical laboratory should
be followed.
Inappropriate collection time and other
adverse conditions often lead to confounding
or uninterpretable data. If there is a diurnal
effect, it is prudent to pool samples or to collect
them at the same time of the day. The initial
survey of healthy and patient samples provide
a rough idea of biological variability. The clini-
cal question is the comparison of the treatment
versus placebo. Appropriate clinical (placebo
and/or predose samples) and assay control
(sample control or QC) data can be assessed
for analytical and biological variability, to pro-
duce unbiased clinical answers. However, for
cancer studies, placebo or baseline samples may
not be available to provide data to parse out
the true drug effect versus the biological and
assay variability.
Reference standard: the
basic yardstick
The basic requirements of reference standards
hold true for both PK and biomarker assays. The
standard is required to be:
Figure 2. Concept of t-for-purpose method validation of biomarkers at various
development phases.
Method development and validation:
Reference standard
Relative accuracy and precision
Sensitivity, selectivity and specificity
Stability
Quality and sample controls
Pre-analytical
sample integrity
Define purpose of study and
biomarker measurements:
Which biomarker(s) to be
included in the study?
Exploratory or advanced
application?
Choose the right
biological matrix and
collection time for
biomarker assay
Method validation & application of protein biomarkers | Review
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n
Puried and well characterized
n
Representative of the analyte in the unknown
samples
n
Available in a large quantity to support the
development program
n
Stable under the dened conditions
n
Accessible to the participating laboratories
Small-molecule biomarkers are well dened
and pure reference standards can be procured
in large quantities to meet these requirements.
Absolute, denitive quantication methods can
be developed and validated, similar to those of PK
assays [12]. Examples are the regulatory peptides
(e.g., insulin), steroid hormones and metabolites.
However, since most biomarkers are large pep-
tides or proteins with molecular weights greater
than 5000 Da and generally heterogeneous in
nature, reference standard characterization and
procurement can be challenging.
Biotherapeutics may also be heterogeneous.
The reference standards are puried and char-
acterized extensively by physicochemical and
biological methods. For example, intact molec-
ular weights are determined by SDSPAGE or
MALDITOFMS. The primary structure of
the protein is assessed by LCMS peptide map-
ping and Edman degradation. Higher order
structures are dened by Fourier transfer infra-
red spectroscopy, near UV circular dichroism,
uorescence spectroscopy and surface plasmon
resonance. Surface hydrophobicity is dened by
aniline naphthalene sulfonate binding. Thermal
stability and stressed data are obtained from
differential scanning calorimetry and dynamic
light scanning. Potency is dened by the specic
cellular bioactivity of the drug. Specications
are dened to assure lot-to-lot reproducibility.
Storage and shipping conditions and boundaries
are also specied to assure stability. Aggregation
is detected by differential scanning calorimetry,
size exclusion LC, SDS, capillary and isoelec-
trofocusing electrophoresis and analytical
ultracentrifugation. Storage degradation is
detected by peptide mapping and SDSPAGE.
Documents of the characterization and stabil-
ity of a standard, such as a certicate or record
of ana lysis and stability, are available to the
bioanalytical laboratory.
Protein biomarker reference standards rarely
meet the requirements of drug compounds; often
the standard is impure, poorly characterized, not
fully representative of the endogenous analyte or
available only in limited quantities. Generally, no
document of certication is provided from either
internal or commercial suppliers. It is doubtful
that the same kind of extensive characteriza-
tion for biotherapeutic reference standards will
ever be used for a protein biomarker unless it
has achieved qualication or surrogacy [27,28].
In addition, the reference material may differ
substantially between lots and manufacturers,
which is a major problem contributing to data
inconsistency [29,30]. This issue must be addressed
with a collaborative effort from pharmaceutical
and diagnostic manufacturers in the future.
Many biomarker standards are obtained from
recombinant expression in noneukaryotic cells;
they may differ from the endogenous forms in
immunoreactivity and bioactivity. The recom-
binant reference standard serves as a relative
yardstick of measurement, assuming that the
immunoreactivity of the endogenous form is
proportional to that of the recombinant form
(parallelism) [13]. Thus, such methods provide
relative quantification. If there is no refer-
ence standard or proportionality between the
endogenous form or the reference standard does
not exist, the methods are quasiquantitative
in nature.
Standard calibrator matrix &
selectivity: matrix effect matters
n Standard calibrator matrix
The preparation of standard calibrators in
a substituted matrix is a major difference
between therapeutic and biomarker ana lysis.
Figure 3. Process of biomarker selection, pre-analytic decisions and method validation to support
drug development.
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Bioanalysis (2009) 1(8) 1466 future science group
Most biomarkers are endogenous compounds
with measurable levels in the biological matrix.
Standard calibrators are preferably prepared in
the intended analyte-free sample matrix [6]; how-
ever, it is difcult to nd analyte-free biological
matrix for biomarkers. The alternative option
is to use a substituted matrix, such as a protein
buffer, a corresponding biological matrix from
another species without the biomarker or to
deplete the biomarker in the biological matrix
by stripping with afnity adsorption or char-
coal. The use of a substituted matrix would avoid
the need for continual screening and testing
of numerous lots of samples to identify blank
controls for standard preparation.
When the calibrators are prepared in a substi-
tuted matrix by spiking a reference standard that
may not be in the same form as the endogenous
biomarker, two types of experiments should be
performed to demonstrate method validity:
n
Comparison of spike recovery from the sample
matrix and the substituted matrix to show that
the concentrationresponse relationships
are similar;
n
Performance of parallelism tests on authentic
samples to show that the endogenous biomar-
ker behaves in a similar immunochemical
manner to the standards.
If the results fail to show similarities, the
method is considered to be quasiquantitative [20].
n Selectivity & matrix effect
Selectivity is the ability of the method to deter-
mine the analyte unequivocally in the presence
of components that may be expected to be pres-
ent in the sample. For small peptides, extrac-
tion procedures similar to those of small drug
molecules can be used to isolate, concentrate and
analyze the peptides by LCMS/MS. A stable
labeled isotope internal standard is added to the
samples to correct for recovery and ionization
variability. For protein molecules, the extrac-
tion step would denature the protein and an
internal standard for LBAs would not be avail-
able. Usually, a simple buffer dilution would be
the pretreatment step, the lack of an extraction
process and internal standard dictates that LBA
specicity and selectivity are solely dependent
upon the ligand-binding reagents [31]. Therefore,
the selection of reagents is of utmost importance
for both biotherapeutic and biomarker LBAs.
With no process to remove matrix com-
ponents, the LBA would be prone to matrix
interferences (the matrix effect). Unrelated
compounds in the matrix, such as heterophilic
antibodies, rheumatoid factor and proteases,
may inhibit or enhance the binding of protein
analytes to the reagents. Often, the immuno-
reactive signal would be suppressed, resulting in
decreased sensitivity and a negative bias.
When carrying out method development for
biotherapeutics, standard matrix curves from
multiple individual lots are assessed for their
performance closeness to a buffer standard
curve. Reagents and incubation conditions can
be manipulated so that the readouts from the
matrix lots converge to those of the buffer curve.
Dilution with a high salt buffer and/or chaotropic
or chelating agent may reduce the matrix effect.
The amount of dilution required to sufciently
remove the matrix effect is referred to as the min-
imal required dilution (MRD) [7]. Since binding
protein types and levels are affected by the health
status and collection conditions, selectivity tests
are conducted by spike recovery at the LLOQ
and at a higher level from at least six matrix lots.
Thus, accurate spike recovery at the LLOQ con-
rms assay sensitivity beyond the single matrix
pool used for standard/QC preparations during
accuracy and precision experiments.
For biomarkers, the matrix effect would also
be tested by spike recovery. However, the basal
levels of the individual lots are determined rst
against the standard curve in the substituted
matrix. Then, the reference material is spiked
into each matrix lot, at a level comparable to
that of the basal concentration. The spike con-
centration cannot be substantially lower than
the baseline and the spiked volume should not
exceed 5% of the individual matrix volume [20].
Spike recovery is calculated after subtraction of
the basal value and compared with the nominal
spike concentration or the mean of the test lots.
If most of the endogenous levels are relatively
high, the LLOQ of the buffer standard would
not be established for the biological samples. As
a result of biological variability, more than the
six lots from various populations required for
biotherapeutics should be tested for biomarkers
(e.g., more than ten from each population) [29,30].
The relative concentrations of the analyte/ inter-
ferent will vary with dose, subject and time point.
Combination therapies may change the amount
of target and binding proteins or bioavailability
of the drug if there is a drugdrug interaction.
One option would be to pool incurred samples
from previous studies of concomitant drugs from
around the T
max
and trough levels and use these as
test samples for specicity and selectivity tests [31].
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n Parallelism
Parallelism is a dilutional linearity test of an
authentic sample. The objective is to show that
the endogenous analyte in the unknown sample,
which may be different from the standard and/
or vary with subjects, behaves similarly, regard-
less of dilution by the standard matrix (or a
substituted matrix in the case of a biomarker).
The experiments are performed for both bio-
therapeutics and biomarkers. However, the
experimental design and results interpretation
are slightly different.
For biotherapeutics, incurred samples from
several subjects are diluted with the standard
blank matrix and analyzed. Therefore, the
experiment can only be conducted after the
in-study commences. Several dilutions are
performed to dilute high concentration study
samples into the standard-curve range for quan-
tication. Each result (the regressed value of the
diluted sample multiplied by the dilution fac-
tor) is compared with the mean of the quanti-
able results and should be within the acceptance
criteria. Although metabolites or drugdrug
interactions may cause nonparallelism, failed
results may be caused by errors from multiple
dilutions of the high concentration samples
rather than real interferences.
For biomarkers, parallelism is an important
component and should be performed during
prestudy validation if possible [30]. Several
individual samples, with concentrations at
the high end of the standard curve from the
initial screening, are chosen. They are analyzed
undiluted and with a dilution factor of three
to four. The ratio of the calculated results
(observed concentration dilution factor)
divided by the mean of the results are plot-
ted against the inverse of the dilution factor.
Parallelism is demonstrated if the ratio is not
affected by dilution.
When parallelism cannot be performed
because samples of sufciently high concen-
tration are not available, dilutional linearity
can be performed in a manner similar to paral-
lelism, using high-concentration spike samples
in place of the authentic samples. The failure
to demonstrate parallelism may mean that the
method is only quasiquantitative [12,29]. In this
case, longitudinal comparison within a sub-
ject would become important, using the pre-
dose baseline as a reference point. The clinical
study design may need to collect more predosed
samples for within subject comparison to the
predose baseline.
Specifcity: the uncertainty of what is
being measured
n Analyte versus structurally
similar molecules
Specicity is the ability of the assay to distin-
guish between the analyte and other structur-
ally related components. Crossreaction with
assay-binding reagents from structurally similar
molecules, such as metabolites, would lead to
overestimation. In contrast to small molecules,
the catabolic species of macromolecule drugs are
not always known and are not puried for the
investigations into their biological activity or
assay interference. It is often assumed that the
intact structure of a protein is required for the
pharmacological action; however, this may not
be true for novel biotherapeutics.
For denitive quantitative methods for small
peptides, the metabolites can be identified,
puried and tested for pharmacological bio-
activity. Puried metabolites can be tested for
crossreactivity against the standard curve. The
concentrationsignal relationship of a LBA is
nonlinear and often the magnitude of metabolite
interference is not monodispersed over the entire
assay range. The estimate of interference is not
as straightforward as that of a chromatographic
method, which uses a single percentage interfer-
ence factor to cover the entire range. Usually,
percentage crossreactivity is expressed as the
ratio of midpoint concentration of the bind-
ing curve of the standard versus that of a given
metabolite (ED
50
). In addition, QC samples
should be spiked with the known metabolites
to conrm specicity [32].
For most protein therapeutics, it is difcult to
ascertain the metabolic species, due to hetero-
geneity; this is even more difcult for biomark-
ers. Multitudes of isoforms and truncated pieces
that can cause specicity problems may exist in
the matrix. Ligand binding coupled with MS
has been used as a novel approach for identify-
ing the truncated forms and guiding method
development for LBAs [33].
For biomarkers, structurally related molecules
include the dosed drug, the precursor molecules
and homologs of the same family [29,30]. In con-
trast to drug assays, the goal of specicity tests for
biomarkers is not to demonstrate absolute speci-
city. Instead, the intended purpose is to provide
information regarding what is being measured
for proper data treatment and interpretation.
There is no specic guideline or consensus
in the pharmaceutical sector on how speci-
city and selectivity experiments should be
Review | Lee
Bioanalysis (2009) 1(8) 1468 future science group
conducted in method validation. The National
Committee for Clinical Laboratory Standards
Working Group denes interference as being
from a known source and the matrix effect
as being from an unidentif ied source [34].
Discussions on selectivity, specicity and free
versus bound tests for LBAs have been occur-
ring at the AAPS meetings organized by the
Ligand Binding Assay Bioanalytical Focus
Group [12,31,35]. LCMS methods can be used
as an orthogonal technique to conrm speci-
city for an LBA method, as well as to detect
differences in isoforms of a biomarker in disease
populations [3640].
n Free or bound target biomarker
to biotherapeutics
Many protein drugs bind soluble ligand tar-
gets. Depending on the binding kinetics, free
and bound forms of the biotherapeutics and the
target ligand coexist in the biological samples
at very different levels. It is necessary to know
what the method should be measuring in order
to conrm assay specicity [35].
Proper PK/PD models are based upon avail-
able data for free and/or bound (e.g., IgE and
omalizumab), bound (e.g., VEGF and VEGF-
TRAP) or total (e.g., IL-6 and canakinumab)
forms [4144]. Knowledge of the free drug levels
is preferred since it reects the species that is
biologically active in vivo. The ligand may exist
in a soluble form in the plasma at high (e.g.,
IgE) or low (e.g., IL-6) abundance. If the drug
is a monoclonal antibody against an abundant
soluble target, the ligand may cause interference
in the drug assay if the LBA reagent binds to
the same or overlapping epitope. Vice versa, the
presence of a high-concentration drug would
interfere with the target biomarker assay.
Depending on the mechanism of action,
the driver of the PD effect can be the free
biomarker or the drugreceptor complex, for
which data would be desirable. In addition,
the total concentration of the target may pro-
vide information on possible compensatory
rise due to induction or membrane shedding.
However, issues of protein binding for bio-
markers and the relevant PD data required
have rarely been discussed, due to the lack of
adequate analytical tools to provide data for the
thorough understanding of the physiology and
binding kinetics.
In the case of small-molecule biomarkers, an
extraction method using organic solvents or a
solid phase can dissociate protein binding prior
to LCMS/MS ana lysis. The method would
provide total (free plus bound) quantication
of the small biomarker.
For a LBA of biotherapeutics and protein bio-
markers, multiple congurations present options
to measure different forms of the drug and tar-
get. The data are valuable for the understand-
ing of binding kinetics. For example, free and
total drug can be measured by the appropriate
choice of binding reagent, coating density of the
capture reagent, incubation time, buffers and
sample dilution. In addition, alkaline or acidic
pretreatment can be used to dissociate drug
ligand binding and then be neutralized before
LBA ana lysis for an assay that measures total
ligand [45,46]. Since it may be difcult to mea-
sure the free biomarker, a consistent method for
measuring the total or bound form may be a
pragmatic option. These techniques and applica-
tions to PK/PD at different drug-development
stages are being discussed by a work team in
the AAPS Ligand Binding Bioanalytical Focus
Group in preparation of a manuscript.
Accuracy, precision & assay range:
quantifcation characteristics
To ensure data quality, assay performance is
evaluated during method validation with valida-
tion samples (VSs) and monitored during sample
ana lysis with QC samples prepared by spiking
known amounts of reference standard into the
biological matrix. VSs are used in method vali-
dation to dene intra- and inter-run accuracy,
precision and sample stability. The prestudy
validation accuracy and precision data of the VS
demonstrate the suitability of the standard curve
assay range and performance characteristics for
its intended application. QC samples are used for
run acceptance during sample ana lysis.
Accuracy and precision experiments and
acceptance criteria for macromolecule drugs
were discussed at the AAPS FDA-sponsored
workshop [9]. Briey, results from multiple runs
of VS over the entire span of a standard curve
would establish accuracy and precision, the
LLOQ and the ULOQ. Total error is the sum
of the systemic error (bias from nominal value
or percentage relative error [%RE]) and random
error (imprecision or percentage coefcient of
variation [%CV]). In-study run acceptance cri-
teria are set based on the total-error information.
At least two thirds of all QC results for a run
should be within a specic percentage (e.g., 20%
for most LBAs) of the nominal values, with at
least 50% accepted for each QC level. A 4-6-X
Method validation & application of protein biomarkers | Review
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rule was proposed for LBA: four out of six QCs
(three QC levels, each in duplicate) should be
within x%, as determined by method validation
total error.
For most biomarkers, since the VSs are pre-
pared by spiking the reference standard into a
pool of authentic matrix with unknown concen-
tration, the nominal values are not known. An
initial target mean can be determined from a few
runs and used to monitor the assay trend with-
out a rigid acceptance criteria. The true value of
the QC may be determined after multiple runs,
using an approach similar to the Westgard Rule.
For relative or quasiquantitative methods, there
is no accuracy assessment and, therefore, the
random error component (%CV) of the assay
is more important.
n Sensitivity: low limit of assay range
Sensitivity for drug ana lysis is determined by
the LLOQ, which is the lowest concentration
demonstrated to be measurable with accept- with accept- with accept-
able accuracy and precision, during method
validation. Extrapolation beyond the LLOQ is
prohibited [6].
Sensitivity is often dened by the limit of
detection (LOD) for diagnostics kits, which is
the lowest amount of analyte in a sample that
can be detected with 95% condence intervals
or other stated probability [47]. For exploratory
biomarkers, variability data at the region below
the LLOQ but measurable above the LOD
may be needed. Samples from subjects from
the intended populations are surveyed by the
method for range nding (see later). If too many
sample concentrations fall below the LLOQ, the
method is not considered sensitive enough for
the intended application. In addition, a sensitive
method is required if the drug effect is expected
to suppress the level of biomarker. In some
cases, it is tolerable to have some subject samples
below the LLOQ and yet above the LOD. If
these data were to be used, one should be aware
of the higher variability in the LODLLOQ
range and interpret the data with caution. For
example, serum C-terminal telopeptides of type
1 collagen (CTx) is a bone resorption biomarker.
Clinical effects of antiresorptive therapeutics,
such as bisphosphonates and denosumab, on
CTx is expressed as the percentage change of
postdose concentrations over that of predose. A
commercial kit was used to monitor CTx change
for denosumab drug development. The kit
LOD was 0.02 ng/ml without dened accuracy
and precision; the LLOQ of 0.049 ng/ml was
dened with accuracy of -6.6%RE, precision
of 20.1 %CV and total error of 26.7%. As the
method was for advanced application in drug
development, we chose to only report data that
were above the LLOQ and not those above the
LOD [48].
n Assay range & sample dilution
The FDA guidance stated that concentrations
of standards should be chosen on the basis of
the concentration range expected in a particu-
lar study [6]. However, the working range of a
LBA is governed mainly by the binding reac-
tion with assay reagents. Most methods are very
sensitive at picogram or nangram per milliliter
level, while concentrations in a PK study for
many biotherapeutics would be in the micro- micro-
gram per milliliter range. Study samples are
diluted into the working range before the assay.
Sample dilution can contribute signicantly to
assay variability within and between laboratories
[Pandya K et al. Strategies to minimize the errors asso-
ciated with manual pipetting in ligand binding assays
to assure data quality of biotherapeutics. Manuscript
in Preparation]. Three levels of QCs (low, mid
and high) within the standard curve range are
used to monitor accuracy and precision perfor-
mance. There has been no common practice of
how sample dilution should be monitored for
each assay; however, it is prudent to have dilu-
tion QCs in an assay run and a strategy has been
proposed to include dilution QCs in the 4-6-X
approach [Pandya K et al. Strategies to minimize the
errors associated with manual pipetting in ligand bind-
ing assays to assure data quality of biotherapeutics.
Manuscript in Preparation]. For novel biomark- For novel biomark- For novel biomark-
ers, the concentration range, modulation and
biological activity of biomarker variants are not
known. They may vary with health status, time
(age and season) and between individuals (gen-
der, genetics and ethnicity). Biological variabil-
ity should be surveyed in samples from normal
and diseased donors, especially in samples from
anticipated patient populations (e.g., 1020
each) to determine if the assay range would be
appropriate. The data should be compared with
the literature and the commercial kit brochure.
It is not uncommon to see discordant literature
data due to the differences in methods (e.g.,
sample collection, reference standard material,
reagents and assay conditions). Most of the time,
patient levels are unavailable or unreliable in the
research-grade commercial kit brochure; there-
fore, the bioanalytical laboratory is responsible
for carrying out the range-nding experiments.
Review | Lee
Bioanalysis (2009) 1(8) 1470 future science group
In addition, the expected drug effect on the
biomarker concentration should be considered
for the assay range. This is often a challenge
during the exploratory phase, as the extent of
drug modulation is not known. The starting
assay range should aim to cover different levels
of healthy and disease populations and also the
anticipated changes from a desirable drug effect.
The ancillary purpose of the range-nding
experiment is to nd authentic samples of low
and high concentrations to be pooled for use as
sample controls (SCs). The SC concentrations
would be determined during method valida-
tion and monitored during in-study runs. SCs
are useful for stability trending and detecting
performance bias due to reagent lot changes [48].
n Data regression: curve ftting &
data assessment
Standard LBA curves are usually nonlinear with
nonconstant error (heteroscedastic). Most LBA
data can be appropriately tted to four- and ve-
parameter logistic models with weighting fac-
tors. Sufcient nonzero standards (six to eight)
are required to dene the regression function
parameters, with additional anchor points out-
side the range to help dene the asymptotes [9,49].
It is recommended to use the residuals of back-
tted standard values, instead of the correlation
coefcient R
2
, to evaluate goodness-of-t. The
precision prole of the VS data during accuracy
and precision experiments conrms the appro-
priateness of the regression model. The accu-
racy and precision data for denitive and rela-
tive quantication methods are used to assess
the systematic and random error components for
total error. These error components are further
monitored with QCs during in-study.
n Acceptance criteria
The 4-6-X rules are commonly used in PK appli-
cations as acceptance criteria for each in-study
run [7,10,11]. The value of X is usually 15% for
LCMS methods. For LBAs, many bioanalyti-
cal laboratories use a xed value of 20%, while
others use a statistical approach to determine X
based on the accuracy and precision performance
data from method validation [79].
No guidance or consensus has been given for
acceptance of biomarker assays. One major pur-
pose of biomarker application is to distinguish
drug effect (dosed vs placebo and/or baseline)
and disease progression (healthy vs disease). The
gap between healthy and disease, and the desir-
able drug effect, should be considered for method
suitability and in determining acceptance cri-
teria. For example, the change in IL-6 is much
greater for sepsis than for asthma. A more sensi-
tive method and stringent acceptance criteria will
be required in drug development for the latter
indication. During the exploratory phase, accep-
tance criteria may be set according to the initial
method performance. After pilot studies, biologi-
cal modulation and assay variability data can be
used to rene the initial acceptance criteria.
Stability
Stability of peptides and proteins in the stock
solution and the intended biological matrix
should be demonstrated [6]. The analyte may
undergo biological (e.g., proteolysis) and chem-
ical (e.g., oxidation leading to aggregation)
changes. Adsorption to the container-vessel walls
or tubing will result in low recovery. Essentially,
stability should be evaluated during sample col-
lection and handling, after long-term (frozen at
the intended storage temperature) and short-
term (bench-top, room temperature) storage and
after going through freezethaw cycles and the
analytical process.
Stability tests of a biotherapeutic analyte
in biological matrix are conducted on the VS,
spiked with reference standards at low and high
concentrations. For biomarkers, since the SC
reects the authentic samples, it is preferable to
use SC over VS for stability tests. In addition,
the same SC set can be monitored during in-
study runs to produce long-term stability data
for trend ana lysis [48].
Reproducibility demonstrated by sample
control data
The screening and selectivity tests for biomarkers
are more rigorous than those for biotherapeutics,
with more lots of matrix from normal and target
disease populations. In addition, pooled SCs are
used to monitor assay reproducibility, reecting
the authentic samples. For example, SC pools at
high and low levels are aliquoted and their levels
determined during method validation experi-
ments and pilot studies from approximately 30
runs. An acceptance criterion of mean 2 stan-
dard deviation can be used. The SCs are then
used as QCs in all in-study runs, as well as part
of the conformance samples for interlaboratory
performance. The SC data can be a common
thread to compare precision and relative accuracy
among multiple studies by different analytical
laboratories. In addition, SC data can be used to
detect reagent lot variability [48].
Method validation & application of protein biomarkers | Review
www.future-science.com 1471
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Application of commercial kits
Commercial kits for diagnostic use have been
commonly adopted for drug development since
they are readily available. The varieties of kits
range from the well-established FDA-approved
(or FDA-cleared) kits to less-proven for research
use only or for investigational use only kits. As
the purposes of drug development are different
from that of diagnosis, it is not recommended
to directly adopt a kit method for drug develop-
ment without method validation [30]. The valida-
tion experiments should evaluate the reference
material and standard matrix, determine perfor-
mance characteristics, patient range and drug
modulation and set up SCs.
The standard calibrators can be a major contrib-
utor to confounding data in research kit applica-
tion. If there are multiple commercial kit sources,
it is prudent to assay the same set of authentic
samples using various kits for comparison. It is not
surprising to nd that the results are totally differ-
ent from one kit to another because the calibrators
(yardsticks) are of different forms. In addition, the
calibrators from one supplier can be different with
time, due to changes in purication processes and
recalibration. If a bulk standard material in suf-
cient quantity can be acquired from one supplier,
standard calibrators should be prepared in-house,
in the appropriate matrix, to assure calibrator con-
sistency throughout an advanced application, such
as in the example of serum CTx [48]. The bulk
standard material also allows the preparation of
sufcient levels of calibrators with anchor points
for appropriate curve tting with weighting, as
well as spiked QCs for accuracy and precision
experiments to dene the assay range.
The assay range should be evaluated against
the population range and the desirable drug
effect. When the biomarker levels are extremely
low, as is the case with the free soluble recep-
tor activator of NFkB ligand, many literature
results using a research kit reported concentra-
tions below the LLOQ. Therefore, most of the
population baseline values would actually be
assay noise [50].
For research-grade commercial kits, QCs or
authentic sample controls may not be available. It
is the analysts responsibility to set up these con-
trols to characterize assay accuracy and precision
and to monitor assay performance. For example,
method validation using commercial kits for
exploratory and advanced biomarker applica-
tions have been reported for tartrate-resistant
acid phosphatase (TRACP 5b) and serum CTx
for bone resorption, respectively [24,48].
The same basic principle of t-for-purpose
method validation must be applied for the
adoption of commercial kits for PK bioana-
lysis. Again, the responsibility resides with the
bioanalytical laboratory to establish the assay
characteristics and determine run-acceptance
criteria. Moreover, kit comparison and rigorous
specicity tests should be conducted [32].
Multiplex assays
Denitive quantitative methods using LCMS
are capable of multi-analyte assays using the
specic mass-to-charge ratios of each analyte of
interest. For peptide analytes, precursor peptides
(e.g., the prodrug or endogenous propeptide)
and their potential metabolites can be quanti-
ed simultaneously [38,51]. The data handling
of multiple analytes would be similar to that of
conventional small molecules and metabolites.
Multiplex LBA platforms and applications have
been developed for biomarkers. Multiple analyte
proling can be bead-based (e.g., Luminex) or
planar (e.g., MesoScale Discovery) formats.
Multiplexing saves time and requires less sam-
ple volume. A panel of potential biomarkers is
tested during early phase to nd those that indi-
cate drug effect. The disproportionate variable
biological ranges of the biomarker analytes and
nonlinearity of the assays should be considered in
method design and assay development [52]. After
the selection of the few relevant biomarkers, the
decision can be made to use either several single-
analyte methods or a multiplex of fewer analytes
for robust assays in later phases.
Future perspective
The position paper on t-for-purpose biomarker
assay validation briey discussed the differ-
ences between biomarker applications and drug
bioana lysis [12]. Protein drug development and
biomarkers share common bioanalytical tech-
nologies but are being applied for different pur-
poses. This review explains in detail the basic
similarities and differences between assays for
biomarkers and assays for biotherapeutics to
support PK/PD studies.
The intended applications of bioana lysis of
therapeutics are usually well dened in each
study protocol. Method validation and bioana-
lysis are performed in a GLP-compliant labo-
ratory, with clear guidance from regulatory
agencies and consensus recommendations from
position publications. The recent discussions on
biotherapeutics, in relation to their correspond-
ing biomarkers, are total and free analyte assays
Fit-FoR-puRpose
method vaLidation
Process of defning study intent
and establishing with
experimental data whether the
assay performance
characteristics are reliable for
the intended application
Review | Lee
Bioanalysis (2009) 1(8) 1472 future science group
Executive summary
Intended purposes of biomarker & biotherapeutic bioana lysis
n
The purposes are well dened for pharmacokinetic (PK) bioana lysis in a study.
n
The intended applications of biomarkers are more diverse and may not be well-dened.
n
A t-for-purpose approach for biomarker method validation and ana lysis is needed; the rigor of method validation and assay
documentation depend upon exploratory or advanced application.
Pre-analytic considerations
n
Pre-analytic considerations include the choice of biomarkers and corresponding biological matrix, the intended application and method
validation plan based on the need for the specied exploratory or advanced application.
Reference standards
n
The reference standards of PK assays and denitive biomarkers are well dened.
n
The reference standards are not the same as, but represent, the endogenous analytes in relative quantitative methods.
Standard calibrator matrix & selectivity
n
Biomarker standard curves often use a substituted matrix devoid of the analyte.
n
More extensive matrix tests are required for biomarkers compared with biotherapeutics.
n
Parallelism of authentic samples diluted with standard matrix are required for a relative method.
Specicity
n
Method should be specic for analyte versus structurally similar molecules (including precursors and pathway metabolites).
n
Specify if free, bound or total target biomarker or biotherapeutic will be measured by the method.
Accuracy, precision & assay range
n
Accuracy and precision validation data are used to characterize method performance.
n
Sensitivity: limit of detection may be used in addition to the LLOQ with caution.
n
Assay range is extended by sample dilution.
n
Nonlinear curve tting is used for data regression. It is necessary to assess data variability from various sources.
n
Acceptance criteria: 4-6-X rule for biotherapeutic PK; exible for biomarkers, depend upon the pathological modulation and drug effect,
in addition to method performance.
Stability
n
Sample controls are used to reect authentic samples.
Reproducibility demonstrated by incurred sample or sample control data
n
There is no requirement for incurred sample with thorough matrix tests and sample control tracking.
Application of commercial kits
n
No direct adoption; users are responsible for appropriate validation.
Multiplex assays
n
Saves sample volume and time at the early stages.
[35,46]. The information is important for the
understanding of drugtarget interactions and
performing robust PK/PD modeling [4144].
The intended applications of biomarkers
are more diverse than those of biotherapeu-
tics. There are applications at various stages
of novel biomarker development (FiguRe 1a),
which are intertwined with drug develop-
ment [29]. The rough category of exploratory
or advanced application is a wide spectrum
that demands exibility in method validation
rigor. Appropriate experiments should be car-
ried out on sample collection, relative accuracy,
precision, range nding, parallelism, selectivity,
specicity and stability [20]. Biomarker bioana-
lysis and method validation are continuous
processes for accumulating knowledge through
the development of inter-related biomarkers and
understanding proteinprotein interactions
of biomarkers and biotherapeutics of similar
mechanisms. Multiplex assays and other physi-
cochemical methods are evolving to enhance
this knowledge.
The development of companion diagnostics
will open up collaborative opportunities for
the pharmaceutical and diagnostic sectors for
effective development and applications of novel
biomarkers in drug development and prognosis.
Acknowledgements
The author thanks Michael Hall for critical review of
the manuscript.
Method validation & application of protein biomarkers | Review
www.future-science.com 1473
future science group
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ment with any organization or entity with a nancial inter-
est in or nancial conict with the subject matter or materi-
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