Proprietary Cell-Line Development for High-Titer AAV Manufacturing

Posted: 1 March 2024

Ahmed Youssef, Senior Manager USP Process Development, spoke with Cynthia Challener, PhD about cell line development for high-titer AAV manufacturing. Along with other industry contributors, they looked at existing techniques and forward at new trends to accommodate more efficient AAV production.

Establishing proprietary cell lines offers substantial opportunities for achieving high AAV titers, shorter production times, rapid production cycles, higher throughput, simplified downstream processes, less likelihood of contamination, greater flexibility in process scale-up, and lower cost-per-dose. Several approaches can be taken, as follows:

• Isolation of high-producing natural clones for transient transfection or infection processes.

• Engineering of cells for the same processes.

• Development of packaging/producer cell lines that eliminate the need for transfection/infection steps.

The optimal technique will depend on the nature of the AAV vector and intended therapeutic application.

Single-cell isolation and cloning is most widely used today, but much research is focused on the development of packaging and producer cells. Cloning avoids the need for genetic engineering, which can raise regulatory concerns. Genetic engineering, however, enables the development of novel host-cell lines for AAV production with improved performance—as long as full documentation of the process and materials used is maintained. Meanwhile, stable producer cell lines eliminate the need for transfection and typically provide higher AAV titers and quality, reducing cost and complexity, but take much longer to develop. Consequently, clonal cell line development is generally used for early-stage programs, while producer cell lines are used for large-scale manufacturing.

Several challenges exist to cell-lined development, regardless of the method, and a comprehensive approach is needed to successfully create high-titer AAV-producing proprietary cell lines. Access to validated cell lines is just the start. Screening is a lengthy process, and comprehensive characterization difficult, as it may require new assays. The cell type, packaging efficiency, viral genome replication, cell growth and viability characteristics, and stability must also be considered. Often these factors play off one another, requiring management of the benefits and risks, with complexity and tradeoffs varying depending on the specific manufacturing platform and product being developed.

There is disagreement as to whether development of proprietary cell lines has positive implications for the cost of and access to AAV-based therapies. Some believe that commercially available cell lines offered through non-exclusive licensing support expedited pathways to pre-clinical and clinical studies, while others see them representing potential for restriction of access to AAV production due to limitation of innovation at smaller companies and inhibition of sharing/collaboration on research.

Despite these issues, ongoing cell-line development and innovation for AAV manufacturing is expected to continue, with advances having overall positive impacts across the sector. Ascend considers proprietary cell-line development the most promising approach to increase cell-specific and volumetric productivity of AAV manufacturing platforms in a disruptive manner.