Development of a robust and scalable purification process of a herpes simplex-based oncolytic virus

This thesis addresses the two critical challenges for the development of a production process for an oncolytic virus. The first challenge is to design a scalable downstream process with sufficient yield and adequate product quality to meet the requirements of regulatory agencies and the second challenge is the short-term stability during the process and during storage at ultra-low temperature. The study begins with highlighting gaps and introducing current knowledge as well as pointing out the significance of addressing the above-mentioned challenges. The research objectives aim to enhance the purification yield and virus stability utilizing the infectivity of HSV-1 as a readout parameter for properties such as activity and quantity. Methodologically, a series of controlled experiments are conducted using bench-scale chromatography and filtration systems in combination with the latest purification techniques like monolithic anion-exchange columns and particle-based fixed-bed columns. In a set of experiments, stability testing under various conditions is performed and process conditions for two chromatographic steps and a subsequent crossflow filtration are optimized under consideration of virus stability. A further assessment of product-related impurities is carried out with regard to the fulfilment of legal requirements. The results demonstrate the potential to improve the infective HSV-1 yield to 6 - 33% at the end of a three-step process, while reducing the product-related impurities to a certain extent by introducing an enzymatic digestion step. Thus, the optimized process sequence enhances the yield of infective virus particles in two chromatographic steps and the crossflow filtration into a buffer containing a stabilizing agent for prolonged storage. In conclusion, this research contributes to the optimized HSV-1 production process, offering a promising approach towards a scalable and robust downstream production process for an HSV-1-based oncolytic virus.