Standard matrix preparation, biomarker

For a majority of biomarker assays, standard calibrators are prepared in an analyte-free alternative matrix instead of the de facto sample matrix of patient samples. For such methods, it is crucial to demonstrate that the concentration response relationship in the sample matrix is similar to that of the alternate matrix. Spike-recovery experiments with the reference standard may be inadequate to evaluate the matrix effect, as the reference standard may not fully represent the endogenous analyte. Instead, parallelism experiments are performed through serial dilutions of a high-concentration sample with the calibrator matrix. Multiple individual matrix lots (>3 lots) should be tested to compare lot-to-lot consistency. In the instance that the limited amounts of sample are available, apooled matrix strategy can be used with caution as discussed by Lee et al. [15]. The samples can be serially diluted with the standard matrix (standard... 

To evaluate assay performance, a set of VS prepared independently of the standards is used. At least five concentrations VS should be prepared, including the target LLOQ and ULOQ, low QC ( 3 times of the LLOQ), mid-QC, and high QC (—75 % of the ULOQ). Accuracy and precision can be evaluated from the total error (the sum of bias and precision) of VS data from the qualification runs in a similar way as for macromolecular protein drugs [17]. Given biological variability and other factors in biomarker research, more lenient acceptance criteria may be used for biomarker PD than for PK studies. Still, it should be recognized that accuracy and precision data of VS in buffer provide only a relative quantification, which may be quite different from measurements in the authentic matrix. 

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