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1/Dilution factor

Figure 3 Parallelism of three individual matrix lots. Each lot is represented by a separate symbol. The sample was diluted with the standard diluent at dilution factors of 1.5, 2.5, and 5. The concentrations of the diluted and undiluted samples observed were all within the standard curve assay range. The mean of the calculated concentrations (observed concentration times the dilution factor) of each lot was determined. The ratios of the individual calculated concentration over the mean of the lot were plotted against 1/dilution factor. The data showed that the ratios were all within the range 0.8 to 1.2, which was the defined acceptance criterion of the assay. Therefore, the parallelism test results were acceptable for the test lots.

tions of diluted and undiluted samples observed should all be within the standard curve range. An example is depicted in Figure 3 : The mean of the concentrations calculated (concentration observed times the dilution factor) of each lot was determined. The ratios of the individual concentrations calculated over the mean of the lot were plot against 1/dilution factor. Parallelism was demonstrated for the test lots because the ratio was not affected by the variable amounts of standard matrix introduced by dilution. When parallelism is not possible due to the unavailability of samples with sufficiently high concentrations of analyte, dilutional linearity should be tested in a manner similar to parallelism, except that high-concentration spike samples are used in place of the endogenous samples.

The failure to demonstrate parallelism should be taken into consideration in data assessment and interpretation. For example, it may mean that the values obtained should be treated as quasiquantitative instead of as relative quantitative measurements (Lee et al., 2006). In addition, within-subject comparison on a longitudinal time sequence would be more useful than between subjects or populations, which affects the clinical study design. All of these considerations would be dependent on the a priori goals of the study.

Selection of Critical Reagents in Ligand-Binding Assays For the development of a first-in-class drug compound that targets a novel biomarker, assay reagents will need to be developed for both the drug candidate and the target protein biomarker. In the case of a macromolecular drug, often an analog of the drug candidate may be used as the binding reagent(s). On the other hand, reagents for most off- t arget biomarkers may be available from commercial sources. These reagents may be established assay kits [U.S. Food and Drug Administration (FDA) approved for diagnostic use] or kits for research use only.

For the purpose of characterization of the biomarker of interest, fit - for-purpose method validation should be conducted with sufficient rigor to provide reliable data from multiple clinical studies. This should include assay range finding, accuracy and precision, selectivity, specificity, stability, and robustness (reproducibility) during prestudy method validation. Additional data will be collected from in-study assay performance, change control method validation, and long-term storage stability. Except in the case of FDA-approved diagnostic kits, many of the assay performance parameters will have to be established by the bioanalytical laboratory. For FDA-approved kits, because the application will be for drug development instead of diagnosis, some of the assay performance parameters will need to be established. These often include selectivity against the target patient sample matrices and specificity against the drug compound(s). If the drug is expected to decrease the biomarker to a level lower than the lowest standard of the kit, the method may need to be modified to increase assay sensitivity.

Antibody pairs are typically chosen as capture and detection reagents. In general, the more selective antibody would be chosen as the capturing agent, especially if it is more readily available than the other member of the pair. A tertiary detector antibody can be used that is conjugated to a reporter enzyme such as horseradish peroxidase. Alternatively, a biotinylated detector antibody can be used together with a biotin-binding protein (e.g., antibiotin antibody or an avidin-type protein) conjugated to a reporter enzyme. The sensitivity of an assay can be increased by varying the number of reporter enzyme molecules on the detection reagents, or by using multivalent strategies to increase the effective signal from each analyte captured.

Some assays use receptors or their fragments as binding partners, most often in concert with a specific second antibody. This arrangement can improve selectivity for specific ligands (e.g., a cytokine activated from a latent precursor, or a particular subtype of ligand with distinct binding characteristics from its homologs). The binding selectivity of such reagents can offer added biological relevance to quantification, and can suggest results that might otherwise be obtained only via functional assays.

Critical Reagents Characterization, Consistency, Stability, and

Documentation Characterization of a novel biomarker often involves many clinical studies that last for several years. If the studies are to be conducted within the same company or from a joint program from different institutions, plans should be made to provide consistent supplies of the same reference material and assay reagents throughout the studies. If the materials are from in - house sources, sufficient production of the reference standard and primary reagents should be assured. In addition, multiple preparations of labeled detector reagents should be assessed during method validation. If the materials are from commercial sources, negotiation with the vendor should take place to assure a consistent and sufficient supply of the same batch material, if possible. Often, multiple batches of materials (reference standards and critical reagents) should be tested during method validation. Documentation should be obtained for the stability of the respective lots over the time span of a clinical study. Documentation of chain of custody should be similar to that of a good laboratory practices (GLP) study. For late-phase studies using multiple clinical sites, the use of a central sample repository offers numerous advantages in controlling the process of specimen collection and labeling of multiple biomarker assays.

In-study sample control (SC) charts such as those shown in Figure 4 can be used for trend analysis of assay performance and stability. A big pool of endogenous sample at low, middle, and high concentrations of a biomarker were prepared during method validation and monitored throughout in-study.

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