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The authors discuss a new, rapid immunoassay for the detection of biomarkers.
The use of biomarker immunoassays to support drug-safety assessment studies is rapidly gaining momentum. Supported by regulatory authorities, industry is moving forward with developing new, translational, noninvasive biomarkers.
The Biomarkers Definitions Working Group defines a biomarker as "a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention" (1). The Predictive Safety Testing Consortium (PSTC), led by the nonprofit Critical Path Institute (C–Path), is bringing together pharmaceutical companies to share and validate safety testing methods under the advice of FDA and EMA. The 18 corporate members of the PSTC are sharing internal experiences with preclinical and clinical safety biomarkers in six working groups: cardiac hypertrophy, kidney, liver, skeletal muscle, testicular toxicity, and vascular injury. The goal of the collaboration is to translate findings in animal studies to measurable risks in humans through the use of novel, translational, noninvasive biomarkers.
An ideal biomarker would monitor a protein, enzyme, or metabolite in an accessible fluid (blood or urine) that would allow detection of toxicity before real injury occurs. A milestone was achieved in 2008 when FDA accepted urinary kidney biomarkers (e.g., KIM-1, albumin, total protein, β2-microglobulin, cystatin C, clusterin, and trefoil factor-3) for the detection of acute drug-induced nephrotoxicity in rat toxicology studies.
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).
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.
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).
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.
Bioanalytical Method Validation