Development of a reporter gene assay on an automated platform

Abstract: Biological drugs have the potential to trigger unwanted immunogenic responses in patients which might affect the efficacy and safety of the treatment. Evaluating the immunogenicity throughout the drug development process is therefore crucial. For measuring the activity of a biological drug and antibodies against the drug, cell-based reporter gene assays can be used. The iLite reporter gene system developed by Svar Life Science consists of cells expressing receptors which are specific for the target of interest and trigger a signalling cascade. The signalling cascade leads down to a promoter fused to a reporter gene giving a quantifiable readout. The aim of this project was to develop a reporter gene assay on an automated platform using two iLite reporter gene cell lines which are normally ran as manual assays. Automating the assays could reduce the hands-on time and increase the sample throughput. To automate the assay it must be able to be incubated without CO2. The hypothesis of replacing the pH buffering of CO2 incubation with the use of a medium with HEPES was investigated. Cell lines targeting TNF-alpha have in this project been used as examples of the iLite reporter gene cell lines. One assay measuring the concentration of the anti-TNF-drug infliximab (IFX) have been tested on the automated platform as well as an assay measuring the antibody-dependant cellular cytotoxicity (ADCC) activity of IFX. Assay characteristics such as accuracy and precision have been evaluated. The study shows that the concentration of IFX can be determined using a reporter gene assay on an automated platform. It was also concluded that for the IFX ADCC assay HEPES can not replace the function of CO2. The results indicate that a part of the iLite reporter gene assays can be automated which opens up the possibility of a more high-throughput immunogenicity testing during the drug development process.