Process development for high-titer production of adenovirus devoid of replication-competent particles in suspension-adapted complementing A549 cell culture

Adenovirus is one of the most attractive viral vectors for therapeutic vaccines and gene therapy with the caveat that replication-competent adenoviruses (RCA) can be produced. To remediate this problem, engineered A-549 adenoviral vector complementing cells (SF-BMAdR cells) were previously generated by our organization for the production of E1-deleted adenoviral vectors without RCA. However, the production process remained to be improved for high titer production and scalability, as cost-effective and scalable biomanufacturing processes are critical for commercializing adenovirus-based vaccines and gene therapy. In this study, we first explored the potential of batch and fed-batch culture to increase maximum cell density and virus productivity by evaluating four different commercially available serum-free media and their combinations, and several feeds. A mixture (1:1) of two culture media improved the maximum cell density from 2.8 × 10⁶ cells/mL obtained in the current batch culture to 4.2 × 10⁶ cells/mL, and increased the virus productivity by 70% at a titer of 1.5 × 10¹⁰ vp/mL. The fed-batch culture process, however, did not yield a significant improvement in either the maximum cell density or virus productivity. In contrast, batch culture with one medium replacement not only increased the cell growth but also resulted in an additional 70% improvement in the virus productivity at 2.6 × 10¹⁰ vp/mL. The virus productivity was further increased to 6.3 × 10¹⁰ vp/mL in a 3 L bioreactor perfusion culture infected at 7.0 × 10⁶ cells/mL. This titer is 7.5 folds of the titer obtained in the current process. This study demonstrated the potential for a drastic improvement in the productivity of RCA-free adenovirus in the SF-BMAdR culture process. Furthermore, various processes developed fulfill different operational needs in manufacturing of RCA-free adenovirus to meet the increasing demands for therapeutic vaccines and gene therapy.