Developing an oncolytic Newcastle disease virus production process with EB66 cells

Newcastle disease virus (NDV) has been the subject of extensive research as a potential oncolytic virus for various types of cancer. While clinical studies are ongoing, the manufacturing of high NDV doses on a large scale is challenging. It has previously been documented that Vero cells are capable of producing 2.4 × 10⁸ TCID₅₀/mL in suspension batch production. However, the requirement of higher input doses and the challenging nature of establishing high cell density processes with Vero cells are significant obstacles in the effective utilization of these therapies. In pursuit of enhanced process intensification and the generation of viral vectors at elevated cell densities, EB66 cells have been identified as a highly effective producer cell line. In this study, the characteristics of EB66 cells in regard to NDV production were examined, with particular reference to cell growth and cell-specific virus productivity in batch and semi-perfusion mode. Optimal infection conditions for producing NDV in batch and semi-perfusion modes were identified for cultivation parameters including temperature, protease concentration (TrypLE), and multiplicity of infection. The favorable production conditions were then transferred to different batch processes using a stirred tank bioreactor and an orbital shaken bioreactor. These processes yielded up to 4.2 × 10⁸ TCID₅₀/mL of the NDV LaSota strain with a cell-specific virus yield of 41 TCID₅₀/cell. First semi-perfusion runs resulted in concentrations of 65 × 10⁶ cells/mL and an infectious virus titer of 7.5 × 10⁸ TCID₅₀/mL. Finally, the potency of the produced viruses was evaluated, and a reduction in tumor size in mice after NDV injection was demonstrated. Overall, these results indicate that EB66 cells could be a viable host for producing oncolytic NDV.