ELUCIDATION OF DRUG MECHANISMS
Beyond safety assessment, there are biomarkers used in early drug development to elucidate the mechanism of action of a drug
and provide preliminary evidence of its effect. As the relationship between a drug or class of drugs and a biomarker becomes
better understood, the goal is to develop clinical assays that identify patients most likely to benefit from the drug. These
biomarkers are termed predictive biomarkers. An early example is the development of HER2 as a predictive biomarker for patients
who were likely to respond to Herceptin (trastuzumab). Presently, there are no specific requirements in regulatory guidance
for biomarker quantification. Indeed, the most recent method validation guideline from EMA specifically excludes the validation
of methods used for determining quantitative concentrations of pharmacokinetic (PK) biomarkers from its scope (1). Notwithstanding
the lack of regulatory guidance, most biomarker immunoassays are validated in a "fit-for-purpose" approach.
The quality of the bioanalytical method is determined by the drug development decision being made based on the pharmacodynamic
(PD) biomarker data. Biomarker assays involve unique challenges including potential interferences from endogenous analytes,
poor availability of a reference standard used for preparing the calibration curve, and a frequent need for low detection
levels in biological fluids. The selection of an appropriate platform for measuring biomarkers is, therefore, driven by technology
and study factors. Sensitivity, sample volume requirements, automation potential, and the general availability of a platform
all need to be considered, along with the intended study species, matrices, and sample volume limitations.
Assay development in conventional immunoassay formats, such as 96 well plate colorimetric enzyme linked immunosobent assay
(ELISA), is a time- and reagent-consuming process, due to long assay times and limited flexibility in experimental set-up.
Traditional colorimetric ELISA typically exhibit good overall performance, but may have limitations in terms of measurement
range and matrix compatibility. For example, small animal models using mice or rats often have limited sample volumes. There
is a general trend away from multiplexed assays in favor of assays developed on more sensitive platforms such as the Gyros
that require minimal sample volumes and short assay run times. The Gyrolab workstation and the Gyrolab Bioaffy CD format
enable immunoassays to be performed in columns using nanolitres of sample volume. The Gyrolab CD is essentially a compact
disk with channels and structures incorporated into it, forming a parallel nanolitre analysis system (see Figure 1).
Figure 1: Gyrolab Bioaffy CD microstructure. One microstructure is equivalent to one data point.
The workstation accurately transfers samples and reagents from microplates to each of the microstructures within a CD. Capillary
action is used to introduce liquid into and through hydrophilic channels. There are hydrophobic barriers that hold reagents
in specific locations. At the desired time, the workstation spins each CD at a precise speed and the centrifugal force moves
the liquid through the structure at controlled flow rates to ensure optimal reaction times. There are several different CDs
available, each with a defined sample volume (e.g., 20, 200, and 1000 nL). The automation and unique flow-through design reduces
"hands-on" time and significantly speeds up throughput. Moreover, the system offers a four-log dynamic range (see Figure 2),
thus reducing the need for repeat sample analysis with additional sample dilution. Results can be generated within one hour
from a single CD or the system can be left unattended to run multiple CDs for up to five hours.
Figure 2: Representitive Gyros standard curves for NGAL (top) and TIMP-1 (bottom). Both assays feature a wide dynamic assay
The Gyros platform, therefore, appears to be well suited for limited rodent sample volumes, and we present here an example
of its application for two inflammation biomarkers: Neutrophil gelatinase-associated lipocalin (NGAL) and Tissue inhibitor
of metalloproteinase 1 (TIMP-1). NGAL is a 178 amino acid protein secreted from specific granules of activated neutrophils.
Its synthesis is also induced in epithelial cells during inflammation. Serum NGAL has been reported to be a useful biomarker
for detection of inflammation and tissue damage, including kidney injury. TIMP-1 is another marker of inflammation as an inhibitor
of the metalloproteinases (MMPs). TIMP-1 is a 194 amino acid secreted glycoprotein that is widely expressed in many cells,
such as fibroblasts, endothelial cells, vascular smooth muscle cells, and monocytes. The release of TIMP-1 is considered to
be a modulator of an inflammatory response (anti-inflammatory). TIMP-1 has been proposed as a biomarker for many tissue injuries,
including kidney and vascular toxicity. In rodent studies, NGAL and TIMP-1 have been employed as safety biomarkers to assess
toxicological risk in the early stages of drug development. Their use has been limited by the volume requirements for current
single-plex assays combined with a loss of analyte sensitivity in multiplex formats.
Optimal antibody pairs for each assay were selected during assay development from multiple commercial antibody sources. Subsequently,
a "fit-for-purpose" method was validated following evaluation of minimum required dilution, accuracy, precision, and dilutional
linearity. Both assays performed well within the "fit-for-purpose" validation criteria. The Gyros platform significantly reduced
assay development time, reagent and sample consumption, and the number of hands-on technician hours needed for assay development
and sample analysis (see Table I).
Table I: Timetable for rapid immunoassay development.
Compared to conventional ELISA, the Gyros platform has shown equal or better overall performance, while exhibiting a wider
analytical range and a reduction in matrix interference effects. The Gyros platform affords a broad dynamic range, thereby
requiring only minimal sample dilution. Methods developed to date demonstrate accuracy and precision well within acceptable
limits over the dynamic ranges of a number of assays.
Roger N. Hayes is vice-president and general manager of laboratory sciences, and Mark J. Cameron is senior manager of biomarkers
and immunoassay, both at MPI Research, 54943 North Main Street, Mattawan, MI, 49071.
1. EMA, Bioanalytical Method Validation (July 2011).