Recent advances in upstream process development for production of recombinant adeno‐associated virus

Abstract

Recombinant adeno‐associated virus (rAAV) is rapidly emerging as the preferred delivery vehicle for gene therapies, with promising advantages in safety and efficacy. Key challenges in systemic in‐vivo rAAV gene therapy applications are the gap in production capabilities versus potential market demand and complex production process. This review summarizes current available information on rAAV upstream manufacturing processes and proposed optimizations for production. The advancements in rAAV production media were reviewed with proposals to speed up the cell culture process development. Furthermore, major methods for genetic element delivery to host cells were summarized with their advantages, limitations, and future directions for optimization. In addition, culture vessel selection criteria were listed based on production cell system, scale, and development stage. Process control at the production step was also outlined with an in‐depth understanding of production kinetics and quality control.

Tuning capsid formation dynamics in recombinant adeno‐associated virus producing synthetic cell lines to enhance full particle productivity

“…The productivity and quality of rAAV of GX6A/B cell lines are on a par with those obtained through plasmid transfection and virus infection. [ 17,42 ] For large‐scale manufacturing, further increases in productivity and product quality are desired alongside adaptation to suspension culture. Given that total virus genomes only reached a level similar to or lower than that of capsids, genome amplification and packaging emerge as prime candidates for intervention.…”

“…These rAAV production systems can produce up to 10 5 rAAV particles per cell on an order of magnitude basis. [ 15–17 ] However, the viral vector produced generally has a large percentage (50%–90%) of empty particles without the genome and payload gene. [ 18 ] These empty capsids might contribute to neutralization of the host humoral immune response, [ 19 ] but a high abundance of empty particles has been reported to cause poor liver transduction due to binding competition.…”

High‐yield recombinant adeno‐associated viral vector production by multivariate optimization of bioprocess and transfection conditions

“…With the fast growing pipelines of gene therapy involving rAAVs, and the significantly higher amount of AAV required per dose for systemic administration, industrialization of manufacturing processes that optimize process yield, robustness, and cost of goods are areas of focus in the biopharmaceutical industry. This is underscored by the relatively low yields of traditional rAAV production processes, commonly reported to be on the order of 1 × 10 10 vg/mL to 1 × 10 11 vg/mL, which ultimately drives the price of these life‐altering therapeutics to beyond $800,000 per dose 10–13 . Therefore, without process development strategies to improve process productivity, these medicines largely remain financially inaccessible.…”

Temporal insights into molecular and cellular responses during rAAV production in HEK293T cells

“… 5 , 6 , 7 , 8 , 9 One pivotal challenge within this landscape centers on identifying optimal transfection conditions capable of augmenting the production of high-titer rAAVs containing the desired gene of interest (GOI). 10 , 11 , 12 Ensuring a substantial quantity of fully loaded rAAV particles carrying the GOI also remains a hurdle to surmount. The existing literature exposes a knowledge gap in the exploration of molecular and cellular dynamics related to HEK293T cells during rAAV production, notwithstanding their widespread use in the biomanufacturing industry.…”