The cell-based assay plays a critical part in the release and stability package for large molecules as it provides data on the biological activity of the therapeutic under investigation. To avoid delays in development of the molecule, the design of the cell-based assay must be considered at an early stage. It needs to be based on the mode of action of the drug, which can be challenging when the mechanism(s) involved are not well defined.
For novel therapeutics, the mode of action is usually unique. As a result, the cell-based assay used for release testing is likely to be unique as well. The design of the assay should take into account the fact that it needs to be easily reproducible as the results will lie on the critical path for release or for stability measurements.
The use of “primary” cell lines, for instance, may initially seem the best option for demonstrating the drug’s mode of action. However, due to the often inherent high variability and reproducibility of these cells, it can be difficult to validate such assays and they should be replaced as soon as possible with more stable cell lines and well before late-stage release.
On the other hand, the use of reporter gene constructs can be the platform of choice for a release assay due to the high sensitivity, tight reproducibility, and, even more importantly, the short time required for the assay (a few hours incubation as opposed to 7–10 days for some assays using primary cell lines).
It is also important to consider the transfer of the cell-based assay to a second quality control laboratory. As a CRO, we are often required to develop and optimize cell-based assays on behalf of our clients prior to validation. However, we are most frequently required to transfer in assays that have been developed externally. On the face of it, this would be expected to be a simple process and it is, so long as it is approached in the correct manner.
Transferring a cell-based assay to a QC lab is a test of the robustness of the assay. It is crucial to think about assay transfer prior to final validation of the method and, in particular, prior to the establishment of acceptance criteria. Assay transfer from development to QC needs to be taken into consideration as a crucial part of assay validation. Without such consideration, assays that already have fixed acceptance critieria during development could result in, at least initially, failure to meet the expected values in the QC laboratory.
As the number of assays being performed grows as the scale increases, then more analysts, greater cell volumes, and more equipment will be involved in carrying out the method. Ideally, the performance of the assay should be evaluated by the external QC lab prior to validation, when the acceptance criteria can still be adjusted and critical parameters can still be optimized.
Specific training of analysts on a particular assay is vital and a GMP requirement. Having sufficiently trained staff requires proper planning as part of assay development and transfer.
Finally, the performance of the assay must be monitored on a regular basis. Data is then used to trend performance over time. Changes in suppliers of reagents, reference standard materials, plates, incubators, equipment, analysts, etc. can significantly affect the performance of cell-based assays and should be continuously evaluated and cross-validated. If necessary, system suitability acceptance criteria should be reviewed and adjusted. In extremis, the assay may need re-validation if significant changes in performance are observed.
Relevant statistical analysis of the assay is essential for performing this continuous monitoring and is a topic in its own right.