Christian Doppler Laboratory for Knowledge-based Production of Gene Therapy Vectors

The vision of the CD laboratory is to move the production of rAAV for gene therapy from a cost-intensive, empirically-driven approach to a knowledge- and model-based process development and production. This requires a profound understanding of relationships such as target lines, therapeutic genes or process conditions interacting with one another and influencing the quality and quantity of rAAV. We will expand analyzes that allow accurate characterization of rAAV and contaminants. One of the most important tasks here will be the differentiation between therapeutic DNA-loaded and empty rAAVs, since different cell lines and different therapeutic genes lead to widely varying ratios between these variants. Product quality is currently being checked using post-process analysis. Such analyzes are time-consuming and costly, and require downtimes in the process. They only provide retrospective information and therefore cannot be used for process control. Therefore, sensors are being investigated that record important process parameters during the process and provide information about its course. This allows process monitoring and control, i.e. intervention in the process to ensure the desired quality. This approach not only increases the security of the processes, but also their efficiency. In order to develop a systematic understanding of important parameters and their interaction, different HEK target lines are examined and genome-wide analyzes of the cell response to virus production are carried out. Based on this, strategies to improve the rAAV yield and quality are developed. Scaling up optimized cell lines and processing strategies to production scale is a multi-step, time-consuming and costly process. The smallest scale currently available for the process development of rAAV production is the laboratory scale, which only allows a limited number of experiments due to the relatively high cost of materials. Only a miniaturized process development platform enables an integrated approach to investigate the relationships between process steps or upstream and downstream processing. This is therefore set up for the process development of rAAV and will include all relevant steps of cell cultivation and downstream processing. Finally, an optimized process is developed on this platform as an example and compared with a current process.