Precision, bioanalytical assay

MS/MS) is the standard detector for bioanalytical assays and drug discovery screening, its use for routine assays of drug substances and products is still limited due to its high cost and lower precision. Nevertheless, LC/MS/MS methods are increasingly used for ultra trace analysis or screening of complex samples. Other detection options include conductivity detection for ionic species and electrochemical detection for neuroactive species in biochemical research. 

Given the foregoing discussion of some of the unique characteristics of macromolecules that lead to clear differences in their pharmacokinetics compared to those typical of small-molecule drugs, there is a subset of the entire group of bioanalytical assay validation parameters that are of key importance in support of pharmacokinetics of candidate macromolecular therapeutics. Assuming demonstration of accuracy and precision of sufficient quality for the intended application of the assay (e.g., non-GLP discovery support or GLP toxicokinetic support, as discussed above), the most important characteristics of a given assay in support of pharmacokinetic studies are likely to be selectivity, specificity, and reproducibility for analysis of incurred samples. These are all related to the ability of the LBA to detect and quantitate solely, or as closely as possible to solely, the analyte of interest. 

Small-molecule quantification by automated infusion using the ESI Chip has been demonstrated to be able to provide comparable precision, accuracy and linear dynamic range compared to traditional LC-MS-MS.28 32 A linear dynamic range greater than five orders of magnitude was demonstrated which enabled validation of bioanalytical assays without the carryover limitations of LC-MS.32... 

The fundamental parameters for bioanalytical validations include accuracy, precision, selectivity, sensitivity, reproducibility, stability of the drug in the matrix under study storage conditions, range, recovery, and response function (see Section 8.2.1). These parameters are also applicable to microbiological and ligand-binding assays. However, these assays possess some unique characteristics that should be considered during method validation, such as selectivity and quantification issues. 

Compared to other bioanalytical methods such as high-performance liquid chromatography (HPLC), the methods used to quantitate mAbs often display less precision and a higher between-day variability. In choosing a bioanalytical method it must also be considered that some assays measure the unbound fraction, the bound fraction, or both. When using FACS, only the fraction of the therapeutic antibody that is bound to its antigen on the cells is counted. In contrast, ELISA measures only the unbound fraction in serum that can react with the offered antigen. 

As for any bioanalytical method, the extent of validation for an immunoassay should be related to the intended application of the assay. Thus, if an immunoassay is intended to support rapid screening in discovery R D, the characterization of specificity and the accuracy and precision specifications may be less stringent than if the assay is used to support pre-clinical and clinical development studies. Indeed, an assay for discovery support may be designed to detect active metabolites as well as parent molecule, so that... 

To avoid bias in the evaluation of the actual precision and accuracy of the bioanalytical method, the results of all QC samples assayed within accepted analytical runs should be reported and taken into consideration in the descriptive statistical analysis. Exclusion of values should be considered only in the case of a documented analytical problem (e.g. chromatographic interference) and the reason for the exclusion should be reported. This applies to both the pre-study validation of the method and the study phase itself. 

If not already available, a bioanalytical chemistry method needs to be defined and characterized for the quantification of the lead in physiological fluids. This assay can then support experiments in some of the other scientific disciplines involved in assessing the developability of the lead and, after appropriate validation, the preclinical, nonclinical, and clinical development of a selected drug candidate. For preliminary studies, a bioanalytical chemistry method should be characterized to demonstrate the range of reliable results, the lower and upper limits of quantification, specificity, accuracy, and precision. In addition, evaluations on the matrix to be used (blood, plasma, serum) should be conducted and the stability of the lead in each matrix should be determined. 

The ultimate goal of an assay or an analytical procedure is to measure accurately a quantity or a concentration of an analyte, or to measure a specific activity, as in some assays for biomarkers. However, many activity assays, such as cell-based and enzyme activity assays, may not be very sensitive, may lack precision, and/or do not include the use of definitive reference standards. Assays based on measurements of physicochemical (such as chromatographic methods) or biochemical (such as ligand-binding assays) attributes of the analyte assume that these quantifiable characteristics are reflective of the quantities, concentration, or biological activity of the analyte. For the purpose of bioanalytical method validation, we will follow the recently proposed classifications for assay data by Lee et al. [4,5]. These classifications, as summarized below, provide a clear distinction with respect to analytical validation practices and requirements. 

Assay acceptance criteria for biomarkers do not solely depend on the deliverable method accuracy and precision, as is the general rule in bioanalytical laboratories. Instead, the consideration should be on the assay suitability for the intended applications considering three major factors (a) the intended use of the data during various stages of drug development, (b) the nature of the assay methodology and the type of data that the assay provides, and (c) the biological variability of the biomarker that exists within and between populations. Thus, the first factor helps shape the assay tolerance or acceptance criteria for biomarkers. 

The optimization and validation of immunoassays for immunogenicity (ADA) testing has been described in detail in several publications [9,14,33,34]. In this section, we will describe the evaluation of relevant performance characteristics (validation parameters) that require the most effort. Some of these are different from the validation of traditional bioanalytical pharmacokinetic (PK) methods for macromolecules [35 37]. Precision, specificity, robustness, and ruggedness are determined similarly between ADA and PK methods. However, recovery/accuracy, sensitivity, stability, linearity, system suitability controls, and selectivity are treated differently between these two types of assays. 

It was originally recommended (Matuszewski 2003) that five different batches of blank matrix (biological fluid in their case) should be tested for ME and RE values in order to assure reliable validation of a bioanalytical method. In the later work (Matuszewski 2006) it was further proposed that assay precision and accuracy should be determined in five different lots of a blank matrix (e.g., a biofluid) instead of repeat (n = 5) analyses using a single lot of blank matrix. Then if an isotope-labeled SIS is not available, determination of the slopes of the five calibration curves and their precision, together with use of <3-4 % (CV %) as a criterion, provides a useful guideline for assessment of the presence or otherwise of a significant relative matrix effect. Of course such a procedure is not always possible, especially for environmental samples a recent example of this problem (Stiiber 2004) involved environmental samples (river water). 

In deciding on uncertainty tolerances the fitness for purpose criterion becomes crucially important. For example, when developing and validating a bioanalytical method, if the achievable accuracy and precision lead the analyst to conclude that the current method is not suitable relative to the original assay requirements, the method in its current form might be discarded altogether, used as the basis for further development of a method that does satisfy the specific criteria, and/or retained as a useful method that can be applied to studies conducted in a less demanding environment. 

For certain types of regulatory or forensic assays there may be specific requirements for the absolute recovery required for the assay, e.g. 70-120%, but the bioanalyt-ical guidelines do not impose any specific requirements for recovery other than that it should be shown to be consistent and precise to ensure method reproducibility. Experiments designed to investigate and optimize the absolute recovery in conjunction with the cleanliness of the extract should be apartof any method development activities, and then tested during validation with the objectives described above. 

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