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

TL;DR

This paper describes a new method to produce high-titer adenovirus without replication-competent particles, improving scalability for vaccines and gene therapy.

Contribution

The study introduces a perfusion culture process in bioreactors that significantly increases adenovirus productivity in SF-BMAdR cells.

Findings

A 1:1 mixture of two serum-free media increased cell density and virus productivity by 70%.

Perfusion culture in a 3 L bioreactor achieved a virus titer of 6.3 × 10^10 vp/mL, a 7.5-fold improvement over current methods.

Medium replacement in batch culture further boosted virus productivity by an additional 70%.

Abstract

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.

Figures