Validation of PK and Biomarker Assays
Assessment of validation parameters
FyoniBio has accumulated many years of proven experience and expertise in the validation of PK and biomarker assays and their application to the analysis of clinical samples. Assay validation is performed according to the current ICH, FDA and EMA guidelines in our ISO 9001 certified and GCLP compliant laboratories. Get in touch with us, we will be happy to support you with the validation of your assay.
What is important for the validation of PK and biomarker assays?
The validation of the PK and biomarker assays ensures that the test results are precise, accurate and reliable, and that the analytical method is fit for the intended purpose. Therefore, quality control samples, spiked serum samples and patient samples are used. The following fundamental parameters are evaluated during validation: the calibration curve, accuracy and precision, sensitivity and quantification limit and range, specificity and selectivity as well as dilutional linearity.
A calibration curve is established over a specified concentration range using calibration standards and allows adequate description of the expected drug concentration in the sample. The standards are prepared in the same biological matrix (e.g., serum, plasma) as the study samples by spiking the analyte of interest with known concentrations in a blank biological matrix. A minimum of six standards in addition to the blank sample are required. Each standard is analyzed in replicates. The lowest and highest calibration standards are then used to define the calibration range.
Assay Quantification Range
The assay quantification range is the concentration range of the analyte in which the analyte can be quantified reliably and with acceptable levels of bias, precision and total error. It is defined by the lower and upper limit of quantification, being LLOQ and ULOQ, respectively.
The accuracy and precision of an analytical method is determined using the QC samples. The accuracy describes the closeness of the measured value to the nominal or known true value and is reported as percent of recovery. The within-run accuracy is determined by analyzing six independent measurements of the QC samples in one run. The between-run accuracy is determined by analyzing six independent measurements per run over several days.
The precision expresses the closeness of agreement (degree of scatter) between a series of measurements and is reported as the coefficient of variation (CV). It may be considered at three levels: repeatability, intermediate precision and reproducibility.
The repeatability, also referred to as intra-run precision, is a measure of the precision of the assay over a short period of time which is typically one day or one run.
The intermediate precision is also termed as inter-run precision and accounts for the variation of the assay under various conditions such as different days, analysts, equipment, and batches of reagents.
The reproducibility is occasionally called between-lab reproducibility defines the precision of the assay at different laboratories. This is particularly useful if the analytical method is standardized or is going to be used by different laboratories.
The precision of an analytical method is also determined using the QC samples. The within-run accuracy and precision are determined by analyzing six independent measurements of the QC samples in one run. The between-run precision is determined by analyzing six independent measurements per run over several days.
Assay Sensitivity (Quantification Limit)
The lowest analyte concentration in the sample that can be analyzed with acceptable accuracy and precision i.e., LLOQ represents the sensitivity of the assay.
Specificity and Selectivity
Specificity is the ability of assay reagents to distinguish between the target analyte and other structural similar and potentially cross-reactive components. It is analyzed using the QC samples in the presence of available related molecules.
Selectivity is the ability of a method to determine the analyte in the presence of other constituents that are expected to be present in the sample i.e., matrix components such as serum proteins, lipid, heterophilic antibodies, rheumatoid factor, proteases, and so on. The selectivity is analyzed by spiking known concentrations of the drug into a blank matrix.
Complex Matrix interference represents a major challenge in the development and validation of a sensitive PK and biomarker assay. Our scientists have gained many years of experience over the years in dealing with and solving these challenges.
Dilutional Linearity and Parallelism
Drug concentrations in PK samples from clinical studies typically span a large concentration range, because they include peak levels (e.g., shortly after infusion) and trough levels (before repeated dosing). Therefore, samples often have to be diluted with different dilution factors before analysis and the accuracy of measuring diluted samples has to be included in the validation. Dilutional linearity demonstrates the ability of the assay to produce results with acceptable accuracy and precision following dilution of the samples. Furthermore, it assesses the presence of a prozone effect. Dilutional linearity is assessed using a high concentration of the target analyte spiked into blank serum samples.
In contrast, parallelism is assessed by using patient samples that have a high concentration of the drug, which falls above the assay ULOQ. Theses samples are then diluted to yield concentration that fall withing the assay range. Parallelism analysis helps detecting matrix effects. It investigates whether or not serially diluted patient samples behave linear and in parallel to the calibration curve. A curve of the diluted patient samples that is parallel to the calibration curve indicates no matrix effects.
Robustness deals with small changes of the assay conditions that could potentially affect the performance of the PK and Biomarker assay. It provides an indication of the reliability of the assay results during normal usage. Variation in assay parameters include changes made in incubation time, reagent and sample volumes. Robustness is assessed using the QC samples.
Preservation of the analyte in clinical specimens from collection at the clinical site to laboratory testing is essential to produce reliable and meaningful data. Therefore, the biochemical stability of the analyte in human serum under different storage conditions is investigated in dedicated stability studies.
These include freeze-thaw, bench-top and long-term stability. They are assessed by using stability samples containing the drug and the matrix after storage at e.g., room temperature, 5°C, -80°C or after undergoing repeated freeze-thaw cycles.
Additionally, the stability of critical assay reagents has to be taken into account and should be included in the validation.
System Suitability Test
The purpose of system suitability tests (SST) is to confirm that the analytical method is suitable for the intended purpose on the day of analysis. It is performed for each analysis using predefined acceptance criteria for certain parameters such as signal, recovery, CV.
An important part of the SST are quality control samples (QC). QC samples are used to evaluate the performance of the method and the stability of the analyte. They are prepared in the same biological matrix as the study samples and they are analyzed in replicates.
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PK and Biomarker Assay Validation – Related Content
Have a look at the relevant guidelines describing the principles of bioanalytical method validation.