By Aryo Nikopour, Senior Vice President, Scientific & Technical Services, Nitto Avecia Pharma Services and Ming Li, Ph.D., Principle Scientist and Team Lead in Biopharmaceutical Development, Nitto Avecia Pharma Services
- An assay readiness assessment needs to be conducted. This normally includes a detailed side-by-side comparison of instruments, software, lab reagents, and critical transfer materials to be used in the transfer study. The purpose is to identify the differences between the labs, assess risks, and design the plan that helps to mitigate the risks.
- Analyst training is a critical next step for method transfer. Cell-based assays are technically challenging and capturing all specifics in a protocol may not even be possible. Some procedural details, such as the pipetting practice and plate emptying techniques, are notoriously hard to capture in the method. Therefore face-to-face training by the analysts who already run the assay successfully is always preferred. This allows scientists from both labs to observe each other and to identify minor variations in lab techniques. Having analysts work together also encourages the transfer of historical knowledge and facilitates troubleshooting.
- Next, feasibility is conducted by the receiving lab to establish the method independently. During feasibility runs, trained analysts will use their own cell culture to perform an additional set of experiments until assay acceptance criteria can be established. Also, successful feasibility runs will demonstrate the equivalency of labs, techniques, equipment, environment and other factors that were identified as risk factors in the initial readiness assessment.
The design of the transfer protocol and scope of work involved in a transfer study could be very different depending on the historical performance of the method as well as the development stage of the product.
Blinding reduces bias between sending and receiving labs
Comparative testing is commonly used for cell assay transfer. For example, both the sending lab and receiving lab can analyze the same set of routine samples and stress samples. These are prepared by the sending lab and blinded to the receiving lab. The acceptance criteria include the precision and percent difference requirements between the two labs. This helps identify if there’s any bias in the results between the sending and receiving labs.
A comparative analysis study is next performed by the receiving lab to generate results at a nominal potency level as well as upper and lower range potency levels known for an assay. The acceptance criteria for precision and accuracy should be statistically derived from the sending lab’s historical data. This demonstrates that the receiving lab can perform the assay and generate results consistent with those from the sending lab.
Performance monitoring identifies variability
After successful validation or transfer, the method can be implemented for GMP sample testing. However, the work doesn’t end there. For every cell-based assay, it’s critical to monitor the performance on a routine basis. This can be done by generating a control chart to track different assay parameters over time. Key parameters such as relative potency trending help to understand the overall variability of the assay and identify any drifting of assay performance. Trending curve parameters such as slope, asymptote, and EC50 values will help to establish equivalence margins required for curve similarity analysis. Also, trending in cell viability and cell doubling time enables early detection of any stress signs from the cell cultures, preventing potential assay failure.
In addition to routine monitoring of assay performance, critical reagents such as reference material, cell banks, and growth factors that support cell culture will need to be qualified, and any new lot of material needs to be bridged to existing lots prior to use for routine sample testing.
Analyst training builds consistency
Unlike other analytical methods, the typical cell-based assay involves extensive manual manipulation of cells and reagents. This combined with multiple steps in sample preparation makes an analyst a major source of assay variability. To maintain stable performance of the method, new analyst training and qualification must be implemented. The training program should involve basic cell culture maintenance, as well as practicing the assay and passing the assay acceptance criteria consistently. A requalification run is needed if the analyst has not performed the assay for a long period of time.
In vitro cell-based assay is an indispensable tool to quantify biological activity and support potency tests for biotherapeutics. Make sure transferring assays to a GMP environment does not become the missing link in your drug development program.