Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5703_Библиотеки_им_академика_М_И_Перельмана
.pdf
inversely proportional to the signal generated. Limitations of this method include radioiso-
tope usage, sample volume requirements (50e300 mL) except for ul trasensitive assays, limited
providers of labeled reagents and instruments (gamma counters). Among the advantages are
the low detection limits obtained as a result of the sensitive detection capability of the radio-
label, and the need for only one high-affinity, high-selectivity antianalyte antibody, which in
the case of short peptide drugs provides a clear benefit over the immuno-sandwich methods
involving both a capture and detection antibody with noncompeting epitopes.
The ECL method was originally developed in a bead-based format by IGEN International/
Bioveris Inc in the 1990s. IGEN/Bioveris discontinued business in 2004 and a plate-based
format from Meso Scale Discovery (MSD) has become the major ECL platform (Meso Scale
Discovery, 2024). The assay utilizes special carbon surface plates, which, according to the
manufacturer, can contain up to 100 different capture reagent spots per well, although
most multiplexed applications do not exceed parallel analysis of 10 analytes per well. The
ECL signal occurs via a ruthenium (Ru)-labeled detection reagent, which luminesces upon
electrochemical stimulation as charge is applied to the plate. The light signal is emitted at
620 nM wavelength, elim inating issues with color quenching from sample matrix. The
FIGURE 5.8 Immunoassay formats for mAb ligand assays including free, total, and bound assays. (a) A free
ligand assay employs a strategy where either the capture or detection antibody competes with the drug antibody for
recognition of the ligand thus only creating a signal when ligand is not drug-bound (i.e., free). (b) In a total ligand
assay antibody pairs for capture and detection need to be identified, which show minimal interference from drug
molecules bound to the ligand: the assay detects both free and drug-bound ligand (i.e., “total” ligand). (c) By contrast,
a drug-bound ligand assay makes use of the ligand and the drug bound to it as molecular handles to capture and
detect. The anti-ligand antibody thereby needs to be selected carefully to again avoid interference from the drug.
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics156
I. Development of biopharmaceuticals
emitted light signal is captured by a sensitive photodetector allowing for a wide dynamic
response range in the assays. ECL assays are primarily used for drug, ADA, target engage-
ment, and other biomarker assays. ECL assays require small sample volumes (<25 mL), can
be miniaturized, depending on method format include few or no wash steps (homogeneous
format), have improved minimum detection limits compared with traditional ELISA plat-
form assays, and can be used to simult aneously evaluate multiple analytes. Limitations
include potential matrix interference problems if using homogeneous format, high costs of
initial instrument purchase, plates and reagents, and operational risks due to support from
only one manufacturer.
AlphaLISA is a proximity-based Perkin Elmer technology, which uses beads labeled with
specific antianalyte antibody reagents (PerkinElmer, 2024). Analyte mo lecules, if present,
bridge the “donor” and “acceptor” beads triggering the transfer of a reactive oxygen species,
which results in emission of light in a sharp peak at 615 nM wavelength. The AlphaLISA plat-
form is typically used for target engagement and other biomarker assays, but is only slowly
gaining acceptance as major platform for drug assays and immunogenicity evaluation.
Attractive features are the small sample volume, and the homogeneous, in-solution format
FIGURE 5.9 Immunoassay formats for anti-mAb antibody assays. (a) Bridging format is typical for initial
screening assays. (b) Confirmatory assays demonstrate that signal in bridging assay is dependent on drug (mAb) as
shown by competition with exogenously spiked excess of mAb. Further characterization is achieved by determi-
nation of anti-mAb antibody isotype (not shown), and assessment of neutralization capability of anti-drug antibodies:
(c) Blocking of drug-target binding is indicative of the ability of anti-drug antibodies in the test samples to interfere
with the molecular interaction between drug and target, (d) while a cell-based assay includes a demonstration of
functional interference by, for example, blocking an intracellular signaling event triggered by target-mediated re-
ceptor engagement.
Bioanalytics 157
I. Development of biopharmaceuticals
of the capture and detection reaction even though the necessity to couple critical assay re-
agents to beads does not strictly speaking represent a true “in- solution” reaction. Drawbacks
include the light sensitivity of the bead chemistry (donor bead photobleaching), which may
require environmental controls (shielding from light sources) and can be a source of assay
variability. Furthermore, the method can be more prone to hook effects than other platforms
because of the need for the homogeneous assay format.
The Gyros system is a semiautomated, miniaturized immunoassay system, which utilizes
CDs, making use of centrifugal forces to apply samples over miniaturize d capture columns
through integrated micro-fluidics (Gyros, 2024). The system uses standard capture (biotin-
labeled antibodies or protein) and detection reagents (fluorophore-coupled antibodies or pro-
teins). The Gyros platform is used primarily for drug, target engagement, and other
biomarker assays. The main advantages of the system are extremely small sam ple size
(nL), semiautomated assay operations, and potential for high sample throughput, which
also offers the benefit of rapid parallel reagent and assay condition evaluation during the
assay development phase. Limitations include high cost of the instrument and assay CDs,
and extra planning to maximally utilize the single-use CDs.
The surface plasmon resonance (SPR) pla tform utilizes special biosensor chips, which have
a variety of special matrices bonded to a gold surface (Biacore, 2024). An analyte or target
protein is bound to the chip surface and then another interacting protein or analyte of interest
is flowed in a solution across the chip. Binding or dissociation of molecules in solution with
molecules on the sensor chip surface causes changes in the refractive index of the chip sur-
face, which is detected in real time by changes in reflection of polarized light at the bottom
of the sensor chip. SPR is widely used to quantitate affinity (on-rates, off-rates, binding con-
stants (K
D
)), specificity, and kinetic behavior of antibodies and antigens. SPR is also widely
used for ADA dete ction and characterization (e.g., isotyping; follow-up characterization of
ADA affinity). The system provides label-free detection of analytes in buffer or matrix envi-
ronment, and depending on instrument configuration can be operated in a high-throughput
mode to allow processing of nonclinical or clinical study sample s. Considerable expertise is
required for optimal SPR assay development and operation. The platform is particularly sen-
sitive to matrix problems and assays require careful configuration. In addition, the instru-
ment and biosensor chips are expensive.
A variati on of the classical immunoassay sand wich format, which is gaining momentum
and finding applications in particular in the area of soluble serum markers is the introduction
of modified detection antibodies to increase assay sensitivity through incorporation of DNA
tags that can be amplified by polymerase chain reaction (PCR). Initial attempts to capitalize
on the amplification of a detection signal through such a hybrid antibodyeDNA assay re-
agent faced significant hurdles regarding reagent stability. Advanced linker chemistries
solved these issues and made this technology more broadly accessible and commercially
available, resulting in immunoassays with a wide dynamic range and up to a 1000-fold in-
crease in sensitivity (e.g., IMPERACER technology from Chimera Biotec GmbH) (Niemeyer
et al., 2007 ; Adler et al., 2008; Chimera, 2024). These immuno-PCR assays have found appli-
cations in biomarker quantitation and immunogenicity assessments, where improved assay
sensitivity directly or indirectly (e.g., through the ability to perform sample dilutions and
thus reduce possible assay interferences) is of benefit over traditional approaches (Spengler
et al., 2009; Singer et al., 2009; Huber et al., 2010). A further extension of this concept has
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics158
I. Development of biopharmaceuticals
been commercialized through Olink (Olink, 2024). Their Olink IMMUNE RESPONSE plat-
form adds improved selectivity to the advantages of increased sensitivity and low sample
volume requirement by introduction of a proximity requirement of two epitopes, which bring
nucleic acid strands on two distinct antibodies together to generate the basis for a PCR-
amplifiable signal (Lundberg et al., 2011 ).
The field of immunoassay technology is in a constant state of evolution and is continu-
ously improving existing immuno-assay methods to increase specificity, sensitivity, reduce
volume requirements, shorten run-times, and increase multiplex analysis. Advances in these
technologies and platforms translate into commercialization for the benefit of the biopharma-
ceutical industry (Nowat zke et al., 2011; Mikulskis et al., 2011; Myler et al., 2011; Lee et al.,
2011).
LC-MS
Liquid chromatography coupled to mass spectrometry (LC-MS) is the most widely used
analytical method for quantitation of small-molecule pharmaceuticals. LC-MS combines sep-
aration of the molecule by liquid chromatography with identification by mass spectrometry.
Liquid chromatography separates and isolates molecules based on their physical/chemical
properties such as size, hydrophobicity and charge (pI). Mass spectrometry identifies mole-
cules via mass/charge criteria using peptide markers specific for the molecule (signature
peptides).
There are a wide variety of LC-MS methods, ranging from simple direct assays with min-
imal sample preparation to complex procedures with multiple sample extraction, isolation
and enrichment steps (Fig. 5.10)(Nowatzke et al., 2011; Campbell and Le Blanc, 2011; van
den Broek et al., 2008; Ezan et al., 2009; Damen et al., 2009). Peptides and smaller proteins
(<10 kDa) can often be analyzed directly without enzymatic digestion and with minimal se-
lection procedures, especially if high sensitivity is not required. However, enzymatic diges-
tion plus extraction/isolation/enrichment steps may be needed for larger therapeutic
proteins and mAbs, or if high sensitivity is required (<nM). Typical steps for LC-MS analysis
of larger therapeutic proteins and mAbs include enzymatic digestion of proteins, extraction/
isolation of signature peptides, enrichment of target peptides, injection onto the LC-MS and
analysis utilizing single (SRM) or multiple reaction monitoring (MRM) (Nowatzke et al.,
2011; van den Broek et al., 2008)(Fig. 5.10).
Identification of the signature peptides is a key step in development of an LC-MS method
for therapeutic proteins and mAbs. A purified sample of the analyte protein is digested and
analyzed by high-resolution mass spectrometry. Peptides with a combination of intense
signal and specificity to the parent protein are selected. Selection of signature peptides for
mAbs, which can be limited to sequences from the variable domains or other limited unique
sequences, can be particularly challenging, and good selection is very important to minimize
interference from endogenous molecules. Unique peptides related to modified portions of
molecules (i.e., linker or chemical modification sites), degradation sites, or other areas of
interest should also be identified.
Selection of internal standards (IS) is an important, but somewhat problematic step for LC-
MS analysis, especially for large therapeutic proteins and mAbs. Internal standards may not
be required for identification of biomarkers or characterization of metabolite or alternate
molecule forms. However, they are essential if the assay needs to be validated. Internal
Bioanalytics 159
I. Development of biopharmaceuticals
FIGURE 5.10 Example LC-MS workflows for peptides, therapeutic proteins (TP), and mAbs. Conventional
workflows used for rapid development and analyses requiring nM LLOQ. Immunocapture workflows generally
used when greater sensitivity is required and for larger therapeutic proteins and mAbs. APR, abundant protein
removal; LLOQ, lower limit of quantitation; PPT, protein precipitation; SEC, size exclusion chromatography; SPE,
solid phase extraction; LLE, liquideliquid extraction; UF, ultrafiltration; SIL-IS, stable isotype-labeled internal stan-
dard; SISCAPA, stable isotope standards with capture by anti-peptide antibodies. Adapted from Campbell and Le Blanc
(2011) and Ackermann and Berna (2007).
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics160
I. Development of biopharmaceuticals
standards are required to quantitatively correct for recovery, dilution, degradation, and other
sample losses during the sample processing and chromatography steps, and for evaluation of
ionization efficiency in the MS. Chemically modified versions of analyte proteins with modi-
fied or substituted amino acids, or different species analogs (i.e., bovine or porcine peptides)
have been used as internal standards, but are not ideal since they may have significant dif-
ferences in physical/chemical properties compared with the analyte protein (Ezan et al.,
2009). Stable isotope-labeled standards (SILs) more closely match analyte protein character-
istics. Traditional methods to generate isotopica lly labeled proteins using full chemical syn-
thesis or protein production from labeled media can be time consuming, expensive, and
may not be suitable for screening large numbers of molecules. Newer methods that utilize
synthetic SIL proteins have shown potential (Brun et al., 2007). Most promising is a recent
whole mAb SIL-IS approach, which used a uniformly heavy isotope-labeled common mAb
for quantitating multiple IgG1 and IgG2 mAbs (Li et al., 2012).
In general, LC-MS provides better specificity and selectivity than immunoassays (Camp-
bell and Le Blanc, 2011; van den Broek et al., 2008; Ezan et al., 2009; Damen et al., 2009; G
Chen et al., 2011) because of its ability to differentiate based on small changes in molecular
mass. LC-MS has a particularly valuable role, therefore, in characterization of modified mol-
ecules and determination of degradation and biotransformation products of bio-
pharmaceuticals in vivo (Nowatzke et al., 2011; Hall et al., 2010). Utilization of LC-MS can
provide extremely valuable stability information for protein design early in nonclinical devel-
opment. LC-MS can also complement traditional immunoassay development by character-
izing the forms of the biopharmaceutical in vivo (intact, complexes, metabolites) and
guiding the selection of optimal immunoassay methods to assess the circulating forms. LC-
MS has a wider dynamic range (>103) than typical immune assays (Table 5.3). LC-MS assays
are not subject to typical biomatrix interferences and may offer a flexible, more unive rsal
method for evaluating molecules in different biologic matrices and in the presence of ADAs.
Overall, complex sample preparation, especially selection, cost, and supply issues with
mAbs for immunocapture, challenges with internal standard selection, lowemedium sample
throughput, detection limits still somewhat higher than imm unoassays (Table 5.3), and high
costs of instrumentation have restricted the use of LC-MS for routine concentration analyses.
However, the continuing rapid development of new methods and instrumentation will likely
expand the role of LC-MS, both as a partner with immunoassays, and as a primary quanti-
tative analysis tool.
Assays
Drug expos ure
Drug assays are the primary measure used to assess exposure and characterize PK in
nonclinical development. Drug assays, either immunoassay or LC-MS, are subject to rigorous
regulatory requirements, particularly when used in regulated GLP (Good Laboratory
Practice) or GCP (Good Clinical Practice) studies (Findlay et al., 2000; DeSilva et al., 2003;
Miller et al., 2001; Smolec et al., 2005; US Department of Health and Human Services,
Bioanalytics 161
I. Development of biopharmaceuticals
2022; Shah et al., 1991). Selection of the platform for drug assays requires clear understanding
of the method validation and operational requirements, as well as the desired assay perfor-
mance (accuracy, precision, selectivity, linearity, robustness).
Immunoassays are the most common platform for drug assays of peptides, therapeutic
proteins, and mAbs (Nowatzke et al., 2011; Damen et al., 2009). However, peptides and small
therapeutic proteins (<10 kDa), are also readily measured using LC-MS/MS. Opioid pep-
tides, hormone analogs and antagonists, antimicrobial peptides, and anticancer peptides
have been assayed by LC-MS (van den Broek et al., 2008). Validated LC-MS assays are
also beginning to be used to support clinical studies (Xu et al., 2010).
An important factor in development, validation, and utilization of any drug assay for a
biopharmaceutical is identification of all forms of drug likely to occur in vivo and selection
of which form(s) to measure (Lee et al., 2011; Kuang et al., 2010). Peptides, therapeutic pro-
teins, and mAbs can all interact with endogenous molecules and exist as “free” and “bound”
forms in vivo. MAbs, in particular, can exist as free and mono- and/or-multivalent complexes
with their target ligands. In vivo biotransformation of peptides and therapeutic proteins and
modified mAbs can also generate intact molecules and various degradation products.
Partially degraded forms of peptides, therapeutic protei ns or mAbs may or may not be bio-
logically active, and selection of the appropriate molecule form to measure for PK or PD anal-
ysis requires additional evaluation of molecule bioactivity. Sample collection, storage,
processing and assay steps (dilution, freeze/thaw, incubation times, relative affinity of assay
reagents vs. drug) can also contribute to formation or dissociation of “free” and “bound”
forms.
The intended use of the drug assay data should primarily influence selection of the mole-
cule forms to be measured quantitatively or qualitatively. PK and PD are often assumed to be
directly related to “free” drug. However, safety assessments may wish to utilize “total” drug
to include all in vivo forms (free, partially bound (one arm only occupied), fully bound) and
to assess potential off-target or framework-related activity for mAbs.
Immunoassays can measure free, bound or total drug depending upon available reagents
and assay formats (Lee et al., 2011). During nonclinical development, reagents (antiidiotypic
mAbs, noninhibitory anti-CDR mAbs; recombinant target molecules represented in a physi-
ologically relevant conformation) may not be available to develop free or bound assays. Total
drug assays are the usual default for nonclinical development. Nonclinical safety assessment
studies are usually conducted with doses where the drug is in very large excess relative to its
target ligand or binding partner. In those situations, bound drug concentrations are very low,
and total drug approximates free drug. However, preclinical pharmacology and PK/PD
studies often use a range of doses to determine minimum effective concentrations in activity
or disease models. Careful interpretation of drug exposure based on total-drug-only assays
may be required in pharmacology and PK/PD studies, particularly in disease models where
ligand or other interacting molecule concentrations may differ from normal animals.
LC-MS assays, in general, will measure “total” drug, unless a sample enrichment step in-
volves selection of a different species (i.e., free or bound drug). LC-MS assays can also be
designed to measure specific degraded or transformed forms of the drug by identifying
unique peptides associ ated with the forms. Early utilization of LC-MS to help identify altered
forms may be particularly valuable since it does not depend upon the development of specific
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics162
I. Development of biopharmaceuticals
antibody reagents for altered forms, and can help evaluate the specificity of early nonclinical
immunoassays.
Since drug assays are required from the beginning of a development program through
approval and commercialization, they are often destined to be outsourced. The challenges
of global outsourcing require the drug assay methods be sufficiently robust to perform
well in a variety of laboratories in many countries, and ideally be low cost, which impacts
the choice of assay platform. Supplies of assay reagents also need to be maintained for years
to support the development programs or reagents need to be available globally with sources
ideally secured either in-house or from multiple possible vendors (O’Hara et al., 2012).
Biomarkers: Target engagement
Biomarkers include a wide variety of measures that are used to evaluate the in vivo activ-
ity of a drug molecule (Rifai et al., 2006; Colburn, 2003). Target engagement assays are a
particular form of biomarker assay for biopharmaceuticals, where the direct interaction of
the drug molecule and target is measured. Target engagement assays typically include simple
direct measurement of the drugetarget interaction (mAb: ligand complexes, peptide or ther-
apeutic protein receptor occupancy) but may also include a signaling or activity readout
downstream of the initial interaction. These assays utilize a wide variety of platforms in addi-
tion to immunoassay and LC-MS, including cell-based assays, quantitative real-time poly-
merase chain reaction (qRT-PCR) and flow cytometry. This discussion will focus on
quantitation of direct mAb interactions with soluble targets (mAb: ligand complex assays).
When mAbs bind to soluble targets, free ligand concentrations are decreased and mAb:
ligand complexes are formed. Observed ligand concentrations often increase due to incorpora-
tion of the ligand in the complex with the mAb (see bound ligand profiles in Fig. 5.5) and pre-
vention of the usual fast elimination of the ligand (Lobo et al., 2004; Putnam et al., 2008). Free
ligand can theoretically also be measured as an indication of in vivo proximal activity if the
baseline ligand levels are sufficiently high for immunoassay measurement. However, most
approved mAbs or mAbs in development target ligands (cytokines, hormones, growth factors)
that are undetectable in circulation (<pg/mL concentrations) at baseline (predosing), making
postdose assessment of free ligand levels impossible. An exception is omalizumab, where the
ligand, IgE, occurs in ng/mL to mg/mL concentrations and both free ligand and total mAb:
ligand complex concentrations have been determined (Putnam et al., 2008; Hayashi et al., 2007).
MAb: ligand complexes are usually measured by an assay for total or bound ligand
(Fig. 5.8). The selection of total versus bound depends primarily upon available reagents.
Measurement of total ligand requires antiligand mAb reagents, which bind at epitopes
different to those of the drug. If the reagent and the drug have overlapping epitopes, free
drug in the samples will interfere with assay, resulting in underestimation of the complexes.
Formation of large complexes, which can occur with li gands with multiple binding sites (i.e.,
homodimeric cytokines, IgE), can also cause overestimation of complex concentration due to
increased avidity and enhance d binding to assay reagents. Sample dilution can dissociate
complexes, especially if the mAb affinity for the ligand is low. Pretreatment by acid, alkaline,
or guanidine to dissociate complexes can also be used for total ligand assays, if the ligand is
stable following the hostile treatment (Salimi-Moosavi et al., 2010). Increases in total mAb:
ligand levels have been used for a number of mAb programs to demonstrate target
Bioanalytics 163
I. Development of biopharmaceuticals
engagement and have also proven very useful for PK/PD modeling of mAb: ligand dynamics
and clinical dose projections (Putnam et al., 2008).
Immunogenicity assessment
Assessment of ADAs is an important part of PK and safety evaluations for all bio-
pharmaceuticals. The immunogenicity assessment strategy for a program should be devel-
oped based on both risk assessment for the specific molecule and regulatory guidance
(European Medicines Agency, 2017; Mire-Sluis et al., 2004; Shankar et al., 2007). Publis hed
guidelines exist for validation of immunoassays, cell-based assays, Biacore assays and for re-
agent selection and preparation and data analysis for ADA assays (Mire-Sluis et al., 2004;
Gupta et al., 2007; Shankar et al., 2008; Dodge et al., 2009; Geng et al., 2005; Mason et al.,
2003; Rup and O’Hara, 2007).
Scientific and regulatory guidance recommends a tiered immunogenicity assessment strat-
egy including screening assays followed by confirmatory assays and characterization assays
(neutralizing potential, titer, isotype). Confirmation and characterization assays are typically
used only for samples, which test positive in the screening assays (Mire-Sluis et al., 2004). The
initial screening assay step is the primary analysis used in nonclinical development. Addi-
tional confirmatory and characterization assays are used on a program-specific basis depen-
dent upon the incidence and impact of ADAs. For example, a high incidence of ADAs in
safety studies may warrant additional characterization to confirm if only drug exposure is
compromised or drug activity has been neutralized as we ll, in particular in situations where
no other drug activity indicator such as a PD marker is included as a study endpoint. In gen-
eral, nonclinical studies are well served, with the evaluation of immunogenicity via the use of
bridging format type ADA assays. In these assays any molecular entity that has the capability
of cross-linking an immobilized, plate-bound drug molecule with a labeled drug molecule in
solution (often performed on an ECL platform, making use of proximity concept to minimize
or eliminate necessary wash stepsdsee earlier sections on assay platform/technologies) will
generate a signal (see Fig. 5.9 and also below). The data are reported either as raw signal
count, which is proportional to the strength of the response, or as an ADA titer, which rep-
resents the required sample dilution to reduce the observed signal to baseline levels. A pos -
itive control reagent, typically a polyclonal ADA (see below for additional details), serves the
purpose of assay performance characterization, and can also be used as a relative standard or
calibrator. It is not recommended, however, to report the ADA data from these screening as-
says in absolute concentrations based on such a calibrator as the composition and character-
istics (binding affinity, detection, etc.) are not representative of the actual ADA molecules
present in samples in terms of species, affinity, polyclonal composition, isotypes, induction
of immune response, and other factors.
ADA screening assays are typically developed as immunoassays, but surface plasmon
resonance (Biacore) and radioimmunoprecipitation formats are also used. Screening immu-
noassays are usually developed in either bridge (as outlined above) or sandwich format
(Fig. 5.9). Sandwich format assays test potential ADA positive samples by using drug coated
on plates or beads and an anti-species-speci fic labeled antibody (i.e., anti-NHP mAb, anti-
human mAb). Assay methods are developed to minimize interference from serum compo-
nents and free drug. Samples are determined to be positive, negative, or inconclusive based
on a cutpoint established from analysis of a panel of drug-naïve samples representative of the
5. Novel biopharmaceuticals: Pharmacokinetics, pharmacodynamics, and bioanalytics164
I. Development of biopharmaceuticals
target population (animal, human, healthy, disease state, etc.dsee also specific paragraph
below).
Positive control reagents are a required component of ADA assays for characterization of
the established cutpoint and for assuring assay reproducibility. The positive control reagent is
typically a polyclonal ADA generated against the drug molecule by immunization of a suit-
able animal. MAbs are used when a positive control reagent for the recognition of a particular
specific epitope (e.g., a crucial modification of a therapeutic protein) is desired. For bridging
format assays, the species for the positive control antibody is not critical. However, for sand-
wich format ADA assays, the antispecies antibody used for detection must match the species
used for the positive control reagent. Specific ADA titers are determined for the polyclonal
ADA reagents for preclinical evaluations and the reagents are used directly as unpurified
serum or minimally purified. These polyclonal reagents will also likely differ from actual
study samples in terms of their affinity, avidity, isotype, epitope specificities, and concentra-
tion. Furthermore, this type of generic polyclonal reagent has utility for preclinical screening,
but is usually not sufficient for screening or characterization of human ADA responses where
specific isotypes, ranges of af finity or multiple epitopes may need to be assessed, particularly
with modified peptides, therapeutic proteins, or mAbs.
Cutpoints for assays are determined by evaluating a large panel of representative animal
or human samples (healthy naïve animal/human samples, as well as representative disease/
disease model samples) and establishing an acceptable false positive rate. The acceptable false
positive rate may use the criteria for human assays (5% false pos itive rate; calculated based on
the mean signal plus 1.645 SD representing the 95th percentile of normal distribution) or
other criteria based on the immunologic risk of the drug and the assay methodology (DeSilva
et al., 2003; Mire-Sluis et al., 2004; Shankar et al., 2006). In most cases the nonclinical study
design may allow collection of late time points where drug-related assay interferences (see
above paragraphs) are minimized, thus increasing the chances to observe low-level ADA re-
sponses, or providing the opportunity to follow a low-level response that may manifest itself
as stronger reaction in later time point samples.
Assay interference can come from nondrug sample components or from drug. No ndrug
sample interference can be particularly problematic when dimeric or multimeric ligands or
drug-target receptors (i.e., shed receptors) are present. Such ligands or receptors can bind
drug and potentially produce false positive signals in bridging format ADA assays. Various
strategies to remove the interfering components have been reported, including blocking by
other mAbs, affinity fractionation, dilution or modifications to assay formats (Dodge et al.,
2009). Free drug is the most common and problematic source of interference in ADA assays.
For rapidly clearing pept ides or therapeutic proteins, the best strate gy may be simply to
obtain samples for ADA assessment after drug washout. Other strategies need to be
employed with long half-life mAbs and high dose levels typically used in chronic safety
studies. Sample pretreatment to dissociate drug: ADA complexes and remove excess drug
may be necessary (Smith et al., 2007; Patton et al., 2005). LC-MS methods have also been
used successfully with internal standards and denaturation/extraction steps (Dubois et al.,
2008; Heudi et al., 2008).
In summary, assessment of drug immunogenicity in the preclinical setting is considered to
primarily aid in evaluation and interpretation of drug exposure, and not to be predictive of
expected clinical immunogenicity. Similarly, within the realm of toxicological analysis, the
Bioanalytics 165
I. Development of biopharmaceuticals
Соседние файлы в папке Библиотека им академика М.И. Перельмана