Piramal's Thaw-For-Use Cell Based Assay
The thaw for use cell based assay format offers several advantages in the planning and scheduling of assays and can also provide a more consistent cell response over that of cultured cells. For early phase studies the transfer of cell based assay require a limited time period of cell culture to generate the research master and working cell banks generating approximately 50 vials of each. During the cell culture period cells can be sacrificed to optimise the dose dilutional series that defines the activity of the drug. An eleven points geometric dilutional series with thee points on each asymptote and 5 points on the slope is the preferred format, although other formats also work. In transferring from a cultured to a thaw for use format, the cell density, cell recovery time and drug exposure time were optimised.
A multiple plate format allows for greater randomisation, increased replication and an increase in the number of test samples that can be analysed together. A multiple plate format offers several advantages (i.e. testing stability samples in the same assay) over that of a single plate format where reference and test dilutional series are analysed either in duplicate of triplicate.
The key elements in the validation of bioassays are defined in ICH Q2R1 guidelines, the United States and European Pharmacopeia monographs and the FDA and EMA Bioanalytical guidelines.
For qualification a minimum of six events was performed by two operators in a design that investigated the repeatability, intermediate precision, linearity and the bias (relative accuracy) of the method. Five mock potency samples; 50%, 71%, 100%, 141% and 200% were prepared for these studies
Robustness studies are conducted using Design of Experiment software, in a factorial design that investigates those parameters that may impact the assay system suitability criteria or the relative potency measurement. The factorial design permits key interactions to be identified and will confirm if the allowed timings or cell density ranges are appropriate.
Validation is conducted using Design of Experiment software in a factorial design incorporating the degree of independent runs required to generate the reportable value.