As the AAV gene therapy field continues to grow and mature, historical knowledge and insights from late-stage process characterization will serve as valuable guidance for improving future process development and control strategies. This talk will focus on upstream process characterization of an adherent transient transfection AAV production process. It will cover topics related to experimental strategy, scale down model validation, production robustness, and raw material variability.

This presentation will focus on the producer cell line process for AAV production, the front runner AAV production platform at Sanofi. The talk will highlight how automation can help build a robust cell line generation platform.

Several adeno-associated virus (AAV)–based gene therapies have been approved in recent years and are providing benefits to patients with rare diseases. However, the price point limits their accessibility. The current approach for cost reduction targets every step of the manufacturing process, including plasmid manufacturing, downstream processing, upstream process development, and scale up. Here we will focus on the use of scale-down models, high-throughput optimization methods, and the use of different transfection and cell culture additives to increase volumetric titers and obtain a cost-effective process for the development of AAV-based therapy for lipoprotein lipase deficiency.

Krabbe disease is caused by mutations in the gene encoding the lysosomal enzyme galactocerebrosidase (GALC), which is essential for normal metabolism of myelin components. Forge Biologics has developed an efficient and scalable process to produce FBX-101 (rAAV expressing GALC) and scaled it under cGMP conditions. In this talk, data will be presented on process development at Forge Biologics to produce drug products for clinical trials for patients with Krabbe disease.

Implementation of a wide range of AAV capsid variants has provided a unique challenge to process development groups, as manufacturing attributes of the AAV differ drastically between serotypes. The Alexion team has developed a manufacturing process with the goal of improving the consistency of the productivity and the quality of AAV produced in the bioreactor, as well as providing flexibility in the purification process to handle performance differences between serotypes.

rAAV technology for gene therapy is rapidly improving, increasing the demand for high yield manufacturing processing techniques. These processing strategies require monitoring of a large number of factors that are all interconnected and impact the overall performance of a run. Multivariate data analysis (MVDA) is a mathematical tool that can be leveraged to generate predictive models of desired outputs such as upstream titer yield.

Adherent Vero cell platforms are well accepted by regulatory authorities for manufacturing of human viral vaccines and therapeutic viruses, but it is labor intensive and costly. To increase cost-effectiveness, we have developed a chemically-defined cell culture media and adapted Vero cells in suspension cultures, which significantly simplifies sub cultivation and process scale-up. For the production of HSV-1, we have obtained 2.7×10⁸ TCID50 mL^-1 in a 3L batch and successfully scaled up to 60L. In a perfusion culture, we obtained 1.1×10⁹ TCID50 mL^-1. We can also produce other viruses, including yellow fever virus and VSV at competitive titers.

Carbon Biosciences is leveraging the genetic diversity of non-AAV parvoviruses to develop novel vectors with increased capacity, minimal seroprevalence, specific tropism and liver-detargeting properties. Carbon has demonstrated successful vectorization of capsids with capacity of 5.5kb and a robust, scalable manufacturing process resulting in high productivity and yield. Carbon platform technology aims to alleviate many of the challenges with current gene therapy and provides optionality for diseases requiring larger genetic constructs.

TruStable Cell Line Technology Platform is a serum-free suspension human cell line engineered for stable rAAV production. This flexible system packages diverse payloads into a variety of capsid serotypes with high titer and exceptional packaging. Polyclonal pools demonstrate productivity at ~8e14 vg/L and ~70% full when measured from crude extracts. Monoclonal derivatives exhibit outstanding productivity, often exceeding 100,000 viral genomes/cell. The TruStable platform is readily scalable for large-scale manufacturing.

High-yield production of rAAV is of pivotal importance yet it remains a critical challenge in current times. Classic rAAV vector manufacturing processes based on transient transfection are limited to low cell density suspension cell culture of mammalian cell lines. By alleviating limitations related to high cell density transfection step and cell culture productivity via process intensification strategies, we achieved improved rAAV production yields with consistent vector quality.

Despite numerous advances in the field of gene therapy, the efficiency and cost of producing rAAV drug products to meet a rapidly-growing industry has significant room for improvement. At AskBio, we have developed a platform-based approach for downstream process development that we believe has resulted in highly scalable and GMP-ready processes. This has accelerated the process development timelines while considerably reducing overall costs of vector production.

Removing empty capsids while ensuring homogeneity, consistency, and stability in the drug substance is the current challenge in the downstream purification of rAAV. This talk will demonstrate chromatographic and non-chromatographic approaches to AAV capsid separation, enrichment, and stabilization. The impact of resin geometry, chemistry, kosmotropic buffer agents, and metal ions will be illustrated. Innovative tools such as mechanistic models and multicolumn chromatography are applied to AAV separation. Using these strategies, we demonstrate > 90% empty capsid removal with a yield of > 80%.

The field of gene therapy has shown the potential to change the paradigm of medicine but faces challenges in the areas of high cost of goods, limited access, challenges to insurance reimbursement, and challenging commercial models, especially for rare disease. This talk focuses on recent advancements in the field and technical development approaches for clinical and commercial success. Topics include emerging technology, development approaches for AAV, program-specific issues, and common hurdles.