A crucial step in drug development, bioassay development can take from three months to a year to accomplish. Time, personnel, and budget resources along with reagent, drug lot, and cell-line availability constrain the process.
Assay reliability is paramount; even seemingly subtle variations can make the difference between a high-performing and a highly variable assay.
Challenges in bioassay development will be the focus of the upcoming IBC “Symposium on Development, Validation and Maintenance of Biological Assays”.
“Ideally, you have responsive and nonresponsive analytical cell banks, key reagents, and several drug lots in place from the start. Additionally, having historical experience with an assay starting from the discovery stage makes assay development and optimization quicker and increases the chances that the assay will run reproducibly when transferred to QC,” says Ken Lewis, Ph.D., principal development scientist, CMC Biologics.
“Frequently, this is not the case and often the analytical tools need to be developed and brought on line very quickly. Depending on the type of drug, for example a biosimilar, there may also be high assay performance expectations from the onset. This makes bioassay development very challenging.”
Trend charts provide empirical evidence regarding the performance of a bioassay and can be useful communication tools, particularly when troubleshooting an assay. To identify key reagents and procedural steps, factors such as reagent lot and cell passage number, and performance parameters such as EC50/IC50, daily maximums and minimums, and slope, are tracked to obtain variance data from multiple plates with multiple analysts on multiple days.
The use of platform assays is another common approach; examples include the CDC and the ADCC antibody assays. With platform assays, many factors are the same; the main difference is the target cell line. This facilitates development and troubleshooting.
Bioassays should be able to detect both hypo- or hyper-potent materials. Although dilutional linearity is used early in the development process to simulate variable potency samples, well-characterized force-degraded samples should be used to challenge the assay and to test the effects of types of drug damage.
Force-degraded samples can be prepared using elevated temperature storage, oxidation, agitation, different pH, etc. to damage the protein. Studies using these samples test not only the potency assay but also other proposed stability-indicating release and characterization assays.
“One of the assumptions for potency assays is that the shape of the dose response is the same for reference material and the test samples. Sometimes a degraded sample may have a mixture of fully active and fully inactive product, merely shifting the response curve to the right.
“In other cases, such as an antibody with two binding arms, if both binding arms are needed for full potency but one has been damaged, binding affinity could change along with the shape of the dose-response curve.
“The potency assay should be able to demonstrate that a degraded test product is no longer behaving the same way as the reference material. Degraded samples, which are unique to every project and dependent on the drug mechanism of action, are crucial,” concludes Dr. Lewis.