Development of robust and quantitative cell-based assays represents a challenging task for any laboratory. Evaluating and optimizing every aspect of an assay early on in the development process will provide more bang for your buck, according to Bhavin S. Parekh, Ph.D., senior research scientist, Eli Lilly and Company (www.lilly.com).
“When companies set up cell-based bioassays they need to know the key limiting factors,“ advises Dr. Parekh. “Optimization is a critical step prior to initiating validation studies. Two of the key parameters that should be optimized are cell numbers per well and duration of the assay. How you change these can greatly affect the signal/noise ratio and the overall quality of the response. In addition, it is also valuable to optimize plate layout, evaluate consumables and reagents, such as plates from different vendors, and different lots of fetal bovine serum.
“But trying to optimize the assay one variable at a time can be a daunting task. Instead it is better to optimize the majority of the parameters via a DOE, or multivariate design of experiment.“
Even the most optimized assay must allow for variations, however. “Biological systems are always subject to change,“ Dr. Parekh says. “Responses may vary depending upon a number of factors. For example, one specific variable, such as the EC50, which is the concentration of therapeutic that provides half maximal activity, can vary considerably with changes in assay conditions and reagents, thus affecting the potency of the sample.
“Finally, the ultimate goal for validating assays is being able to demonstrate the similarity in curve response of sample and reference standard. So, it is important that the reference standard is representative of the final API. Optimizing these and other criteria are key aspects of assay development, especially if the assays are to be used as quantitative tools.“
To obtain optimum performance of cell-based assays, it is critical to employ process controls at each step, says Gwendolyn M. Wise-Blackman, Ph.D., senior manager of cellular technologies, Cardinal Health (www.cardinalhealth.com).
“We are a contract development organization,“ Dr. Wise-Blackman notes. “Our customers all differ as to the extent to which they´ve optimized their assays. Our job is to take their basic method and see that it will carry through to the GMP environment. To do this we implement process controls and look at long-term issues. So, for example, we assess ruggedness, repeatability, accuracy, linearity or parallelism, range, and specificity of assays.“
Dr. Wise-Blackman points out that all steps from simple to complex are analyzed. “We initially establish cell-culture conditions, such as the best vendor, the percent serum, level of carbon dioxide, and even how frequently the incubator door can be opened. Little changes can have big effects. We had one customer who recommended a cell-based assay, yet when we tried it, we got different results than expected. We found that the critical variables were changing culture conditions carefully to limit density, controlling for passage number and the need to systematically subculture cells two days prior to testing. This example illustrates how subtle culture conditions could critically impact results.“
It takes time to map out and optimize assay conditions. “Although it´s fairly time consuming, optimizing conditions early on is the best way to go,“ she says. “Also, we will work with customers to be sure that the cell line chosen is the best for their studies. One wants to choose a cell line for assaying that best replicates the biological mechanism impacted by their product.“