Biological assays, validating

Direct isolation of sufficient quantities of each metabolite for structural characterization, assay validation and pharmacological or toxicological testing from in vivo studies using biological specimens is, therefore, often impossible, particularly from dmgs with a low therapeutic index. Furthermore, many metabolites have structural modifications which are difficult to replicate by traditional chemical methods. A number of synthetic steps may be required to prepare such metabolites from the API, or, in the worst case, a completely new synthetic route may need to be developed. 

It is an essential condition of biological assay methods that the tests on the standard preparation and on the sample whose potency is being determined should be carried out at the same time and, in all other respects, under strictly comparable conditions. The validation of microbiological assay method includes performance criteria (analytical parameters) such as linearity, range, accuracy, precision, specificity, etc. 

The performance of the presented bioanalytical assay method was assessed following the guideline for biological method validation presented by Shah et al. (Shah 2001). 

A valid biological assay to measure the biological activity should be provided by the manufacturer. Examples of procedures used to measure biological activity include ... 

First, it should be remembered that the basic observation, a rise in plasma factor VIII concentration, depends entirely upon a biological assay, in which the corrective effect of the test plasma is compared to that of a control plasma when both are added to plasma obtained from a severely affected hemophiliac, or to an artificial system containing necessary clotting factors other than factor VIII. In acute experiments it has been usual to assay the subject s pretreatment plasma as well as the plasma obtained after the experiment, or to use the pretreatment plasma as the standard (nominally 100%) for the assay. The second procedure eliminates errors due to differences between subjects, but uncertainty still remains regarding the effects of the experimental treatment upon the assay system, apart from a possible true increase in factor VIII concentration. A number of experiments have therefore been directed to testing the validity of the... 

Biological assays are often noisy and laborious. With careful application of experimental design, cell culture bioassays can be made quite accurate and precise. The core information needed for validation can come from two experiments. One experiment studies accuracy and precision followed by a variance component analysis and a summary table that describes the expected performance of the system at various levels of replication. A second experiment uses a minimal fractional factorial design to study robustness, followed by a comparison of confidence intervals on effect sizes with a previously established indifference zone. 

In lead optimization using conventional batch technology, irrespective of whether parallel or iterative mode is chosen, validation and optimization of reactions tends to be one of the major rate-limiting steps. Based on the model described in Fig. 14.5, however, it can be seen that if the biological assay was replaced by a chemical assay and the conditions not the reagents are varied, then an auto-optimization could be carried out. Replication of the optimized channel as a parallel bundle could then provide a means of amplifying the amounts of material generated. 

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