Prestudy Validation Phase

At this stage, it is assumed that the analytical method that is being validated has been developed and optimized, as described in the previous section. Accordingly, 

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Table 4.4 lists the analytical performance characteristics that need to be confirmed during the prestudy validation phase [4 5,9]. In addition, this table highlights the activities that are needed to progress from method development to help ensure the method will meet the prestudy a priori criteria for method acceptance and for successful acceptance of batch runs (in-study validation). 

During the prestudy validation phase, samples should be spiked at the anticipated LLOQ and the ULOQ of the assay. These validation samples should be assayed as part... 

Unlike the prestudy validation phase, where the experiments are relatively time consuming and need to be rigorous, validation rules during the in-study phase should be simple and inexpensive. An in-study rule that is largely accepted in the bioanalytical community is the 4 6 15 rule and is defined in the FDA guidance [3] as ... Atleast four of every six QC samples should be within 15% of their respective nominal values. .. to accept a run. This mle provides a simple and practical guide for routine follow-up. 

During sample analysis before assessing the QC samples for acceptance, the standard curve must be deemed appropriate by predetermined criteria. Only after the curve is accepted may the assessment of QC samples continue. QC sample results determine whether the assay run is valid. Acceptance criteria can be based on 4-6-20 rule or on Total Error and should be predicated on the criteria used in both the development and the prestudy validation phase. Overall, the immunoassay is a highly sensitive assay that can be used to quantify protein and peptide drugs in a biological matrix, often routinely in the pg/mL range. 

Validation of bioanalytical assays in general and LBAs in particular has been the subject of intensive debate for the past 18 years or more. Chapter 4 focuses on the key agreements on a phased approach to the validation of LB As, including evaluation of all critical validation parameters prior to implementation of the method to the analysis of any study samples (prestudy validation) as well as in-study validation to assure high performance of the assay during analysis of actual study samples. Also covered in this chapter are the topics of when and how to conduct full validations, partial validations, and cross-validation. 

Performance Characteristic Method Development Prestudy Validation (Validation Phase) In-Study Validation (Implementation Phase, Sample Analysis Phase)... 

The basic aim when applying prestudy and in-study validation procedures to a measurement method is to reconcile the objectives of the two validation phases. When the tolerance interval approach is used for prestudy validation and the 4 6 2 rule is used during in-study validation, the common objective is to control the proportion n of measurement results (X — jiy) that fall within the acceptance limits [—2,+ 2]. 

The alignment of risk between the prestudy and in-study validation phases can be envisaged in two ways, as shown by Boulanger et al. [29]. On the one hand, if the number of QC samples, n, to be used and the minimum, 5, of QC samples within the acceptance limits in the s n X rule are fixed (e.g., 4 6 15), the value of 7imin should be chosen so as to ensure that if the method remains valid, the s n X rule is accepted in most cases (e.g., with a minimum probability ymin). On the other hand, for a given prestudy validation scheme and X fixed), the value of 5 QC samples within the acceptance limits (for a given n) should guarantee that most of the runs will be accepted if the method remains valid. 

The acceptance criteria used during prestudy validation and in-study validation should be consistent with each other. Lack of consistency can result in a validated method failing more often than expected during the in-study phase or vice versa. To ensure this consistency, either the commonly used in-study acceptance criteria such as the 4 6 15 rule can be modified by increasing the number of QC samples (e.g., 8 12 15 rule) or the prestudy validation criteria based on tolerance intervals can be altered to ensure that a higher proportion of the measured results fall within the acceptance limits. 

The stages of validation of biomarker assays include establishment of the biomarker (development), so-called prevalidation, prestudy validation, and in-study validation [13-15]. The following short discussion will focus on the GLP-like definitive and relative quantitative assays. As the development and validation of an assay for novel biomarkers is quite diverse, the application of strict validation procedures appears problematic. Therefore, upon establishment of the prototype assay in the development phase, a formalized validation plan should be developed that... 

Validation comprises pre-study and within-study phases. During the prestudy phase stability of the stock solution and spiked samples in the biological matrix, specificity, sensitivity, accuracy, precision and reproducibility should be provided. Within-study validation proves the stability of samples collected during a clinical trial under storage conditions and confirms the accuracy and precision of the determinations. 

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