However, certain types of tests should be validated much earlier in the drug development program. If one does a quality risk
assessment, one will find that validation would be required of preclinical GLP tests for product safety and toxicity; of tests
to demonstrate the absence of contaminating microorganisms or to validate virus removal/inactivation processes; and of good
clinical practice (GCP) laboratory analyses of critical factors such as blood levels of the product and its metabolites, or
antibody or other surrogate marker levels in blood and tissues.
"Test verification" is a term applied to analytical methods considered already validated. These methods typically appear in
compendia, such as the US Pharmacopoeia or the National Formulary. A laboratory need only demonstrate its ability to perform
the test according to stated specifications. If standards are available for these tests from bodies such as the National Institute
of Standards and Technology or the World Health Organization, these should be used in test verification. Similar criteria
can be applied to tests developed in-house that have already been validated in the course of other development work. As a
guide, a typical list of types of analytical methods normally expected to be qualified, verified, or validated is shown in
Table 2. An Analysis of Test Requirements
Test Method Validation
To paraphrase definitions given in various guideline documents, "Validation involves documenting, through the use of specific
laboratory investigations, that performance characteristics of the method are suitable for intended analytical applications
and are reliable. The acceptability of analytical data corresponds directly to the criteria used to validate the method."
The critical parameters (or "performance characteristics") for any test method are typically defined by the guidelines documents
as follows: :
Sensitivity: The lower limit of detection (LOD). The lowest concentration of an analyte that the analytical procedure can reliably differentiate
from background "noise."
Specificity: The ability to assess unequivocally the analyte in the presence of components that may be expected to be present. Typically
these might include impurities, degradation products, and matrix.
Precision: The closeness of agreement (degree of scatter) among a series of measurements obtained from multiple sampling of the same
homogenous sample under prescribed conditions. Three levels of precision may be considered:
- Repeatability — precision under the same operating conditions, over a short time period
- Intermediate precision — within laboratory variations; different days, analysts, equipment, etc.
- Reproducibility — precision among different laboratories, e.g., by collaborative studies
Accuracy: The degree of closeness of the determined value to the nominal or known true value under prescribed conditions.
Quantification Range: The range of concentration, including the upper limit of quantification (ULOQ) and the lower limit of quantification (LLOQ),
that can be reliably and reproducibly quantified with accuracy and precision through use of a concentration-response relationship.
Note that LLOQ is not necessarily the same as LOD. It should be understood that the lowest amount of the analyte that may
be detected may not be assayed accurately by the test.
Linearity of the Standard Curve: The extent to which the relationship between the experimental response value (or a mathematical manipulation of this) and
the concentration of the analyte approximates to a straight line. This relationship may also be called the calibration curve.
The linearity of the calibration curve will affect the quantitation range, since departure from linearity in some portion
of the graph will affect the reliability of the assay.
Robustness: The ability of the method to deliver accurate, precise results under normal variations of operating conditions. Such variations
may include performance by different analysts, the use of reagents from different suppliers, or of reagents with differing
storage times. Also, different types of analytical equipment, perhaps with different sensitivity or accuracy, may be in use
in the test development laboratory and in those laboratories that will perform routine quality control (QC) testing. The more
robust the assay, the less the effect of these factors on its accuracy and precision.