Conclusion

This work reports, to our knowledge, the first single-cell RNA-sequencing analysis of Sf9 insect cells during the production of AAV using a low MOI, dual-baculovirus system. The findings of our work highlights production bottlenecks of this system, suggesting the need to reduce cell heterogeneity (e.g., by applying synchronization strategies) and differences in transgene expression (e.g., by applying similar promoters), as well as enhance and/or prolong the production process (e.g., by manipulation of specific pathways) for its improvement. Overall, this study lays the foundation for further single-cell transcriptomics studies in insect cell-based bioprocesses and further elevates our knowledge on the underlying biological mechanism of multi-vector production processes.