Development of a reporter gene assay on an automated platform

English. 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.

English. Biological drugs are drugs that are produced in or derived from living cells, such as bacterial or animal cells. They are used for treating cancer and other diseases. During the treatment, the immune system of the patient might try to fight the drug by for example producing proteins called antibodies that can bind to the drug molecule. The drug might then not be able to treat the disease and the immune response might also cause serious illness to the patient. It is therefore essential to test that the drug is efficient and safe to use before being released to the market. During the treatment, patients can be monitored by measuring the drug level and the antibodies against the drug, which can improve the treatment outcome. One method to measure these levels is to use cells that are genetically modified to detect the specific biological drug of interest. It is called a reporter gene assay. The cells respond to the biological drug and generate a light that can be measured. From the measured signal, the concentration of biological drug and the presence of antibodies against the drug can be determined. Reporter gene assays are normally performed manually which takes up to an hour to do. In this project it was attempted to perform the assay using an automated system where the liquids are automatically handled. This could reduce the time needed to spend on the assay and enable faster development and testing of biological drugs. The assay has been performed manually to first see if the incubation steps, where you wait for the reagents to react, can be done without CO2 which is often used when incubating cells. The assay was then setup on the automated platform to automatically handle the liquids and the incubation steps. It was shown that for one of the assays tested, the automated assay is comparable with the standard manual assay when it comes to accuracy and precision. On the other hand, the second assay tested did not show good result when incubated without CO2. It was shown that this assay can be automated for only a part of the drug levels tested. In conclusion, one of the assays tested during this study could be transferred to the automated system and the other assay is more problematic to automate.