The use of adeno-associated viruses (AAV) as vectors for gene and cell therapy has risen considerably in recent years. Consequently, the amount of AAV vectors required during the validation and clinical trials has also increased. AAV serotype 6 (AAV6) is well-documented for its efficiency in transducing different cell types and has been successfully used in gene and cell therapy protocols. However, the number of vectors required to effectively deliver the transgene to one single cell has been estimated at 106 viral genomes (VG). Overall, this means that large-scale production of AAV6 is needed. Suspension cell-based platforms are currently limited to low-cell-density productions, hindering the potential of this production process to increase yields. Here, we investigate the improvement of the production of AAV6 at higher cell densities. The production was performed by transient transfection of HEK293SF cells. When the plasmid DNA is provided on a cell basis, the production can be carried out at medium cell density without effects on cell-specific titer or particle functionality, resulting in titers above 1010 VG/mL. Medium supplementation alleviated the cell density effect, in terms of VG/cell, at high-cell-density productions. On the other hand, the cell-specific functional titer was not maintained, and further studies are necessary to understand the observed limitations. The medium-cell-density production method reported here lays the foundation for large-scale process operations, potentially solving the current vector shortage in AAV manufacturing.

Plasmid transfection of mammalian cells is the dominant platform used to produce adeno-associated virus (AAV) vectors for clinical and research applications. Low yields from this platform currently make it difficult to supply these activities with adequate material. In an effort to better understand the current limitations of transfection-based manufacturing, this study examines what proportion of cells in a model transfection produce appreciable amounts of assembled AAV capsid. Using conformation-specific antibody staining and flow cytometry we report the surprising result that despite obtaining high transfection efficiencies and nominal vector yields in our model system, only 5-10% of cells appear to produce measurable levels of assembled AAV capsids. This finding implies that considerable increases in vector titer could be realized through increasing the proportion of productive cells. Furthermore, we suggest that the flow cytometry assay used here to quantify productive cells may be a useful metric for future optimization of transfection-based AAV vector manufacturing platforms.

The Vero cell line is the most used continuous cell line in viral vaccine manufacturing. This adherent cell culture platform requires the use of surfaces to support cell growth, typically roller bottles or microcarriers. We have recently compared the production of rVSV-ZEBOV on Vero cells between microcarrier and fixed-bed bioreactors. However, suspension cultures are considered superior with regards to process scalability. Therefore, we further explore the Vero suspension system for rVSV-vectored vaccine production. Previously, this suspension cell line was only able to be cultivated in a proprietary medium. Here, we expand the adaptation and bioreactor cultivation to a serum-free commercial medium. Following small scale optimization and screening studies, we demonstrate bioreactor productions of highly relevant vaccines and vaccine candidates against Ebola virus disease, HIV and COVID-19 in the Vero suspension system. rVSV-ZEBOV, rVSV-HIV and rVSVInd-msp-SF-Gtc can replicate to high titers in the bioreactor, reaching 3.87 × 107 TCID50/mL, 2.12 × 107 TCID50/mL and 3.59 × 109 TCID50/mL, respectively. Further, we compare cell specific productivities, and the quality of the produced viruses by determining the ratio of total viral particles to infectious viral particles

Despite rapid progress in the field, scalable high-yield production of AAV is still one of the critical bottlenecks the manufacturing sector is facing. The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as a mainstream platform for a scalable production of recombinant proteins with clinically approved products for human use. In this review, we provide a detailed overview on advancements in IC-BEVS for rAAV production. Since the first report of baculovirus induced production of rAAV in insect cells in 2002, this platform has undergone significant evolutions. The original three baculovirus system was improved to enhance expression stability and further streamlined to reduce the number of baculoviruses to two and eventually one. The one baculovirus system consisting of an inducible packaging insect cell line was further improved to enhance the AAV vector quality and potency. In parallel, implementation of advanced manufacturing approaches and control of critical processing parameters have demonstrated promising results with process validation in large scale bioreactor runs. Moreover, optimization of molecular design of vectors to enable higher cell-specific yield of functional AAV particles in combination with bioprocess intensification strategies may further contribute to addressing current and future manufacturing challenges.