The Key to Moving Gene Therapy from the Clinic to Commercialization Scaling Up the AAV Production Process

As gene therapy is experiencing a second wave of interest, recombinant adeno-associated virus (rAAV) has rapidly emerged as one of the most attractive viral transfer tools, thanks to its favorable safety profile and long-term transgene expression. The recent approval of rAAV molecules for the treatment of congenital amaurosis and spinal muscular atrophy has been paving the road for the development of several rAAV-based therapeutics targeting a continuously increasing range of indications. To support this rapid growth, the field is in high demand for robust, scalable and economically viable manufacturing processes.

Challenges in scaling up AAV-based gene therapy manufacturing

AAV mediated gene therapy offers the potential for curative therapies for many fatal and debilitating diseases, however the translation of these concepts into licenced products is stymied by manufacturing challenges leading to very high costs and delayed commercialisation. This is an issue which vector developers need to address, specifically around productivities and vector purities including levels of empty non-functional capsids. To date vector developers have focused on the optimising of vector targeting and gene delivery, however there is an increasing need to recognise the issue of manufacturability of vectors, and to be able develop screening and assessment of novel vectors to ensure that in future innovations and development can be produced to the required quality and in the desired quantities and at an acceptable cost.

The production of AAV viral vectors is a complex process that has to meet safety and efficacy requirements while considering cost and market demand. In the upstream and downstream processing flow of scale-up production, the downstream can account for a significant portion of the total cost of virus production, and in order to reduce production costs, an efficient and stably reproducible method to generate high purity viral vectors is required. The main challenges in the downstream workflow include cell lysis, filtration and purification.

The Key to Moving Gene Therapy from the Clinic to Commercialization Scaling Up the AAV Production Process Fig. 1 Upstream and downstream processes for rAAV production. (Selvaraj, et al., 2021)

Production process development: the cornerstone of AAV carrier scale up production

In the long term, we need to increase research into the mechanisms of cell production and how to improve vector productivity and efficiency in the production of fully empty coat vectors. Current means of improvement include mainly through molecular or synthetic biology approaches, cell line selection or modification, or through more traditional approaches around optimization of culture/bioreactor conditions. In addition, there is a need to improve understanding of vector stability through purification operations, all of which are extremely challenging tasks.

Viral vector starting materials

All AAV production processes require starting material, either plasmid or baculoviral/HSV starting viruses. With the prevalence of triple transfection-based AAV manufacture, the plasmid used is crucial. Plasmid generation typically requires a standalone manufacturing process based on microbial expression technology. Focus on quality is required, not only for the quality of plasmid used in the manufacturing process, but also for the impact on the quality of the resultant viral vector produced.

Production of AAV

To realise the full potential of gene therapy products, manufacturing processes are required to produce high-quality vectors, have high productivity, rapid development, and scalability to deliver to GMP, in addition to being able to produce multiple AAV serotypes. Traditionally, cell-based production of AAV has been through the use of adherent systems, with transient transfection driven through calcium chloride methods. Early production resulted in a typical GMP manufacturing campaign requiring.

Purification and full viral vector separation

From an AAV purification perspective, fully locked and standardised platforms are a challenge, especially when considering a multi-serotype approach. Different AAV serotypes present different challenges driven by the capsid structure and associated properties. Advanced manufacturers are using a tailored approach depending on the serotype used, a 'toolbox' approach. The first consideration is often in regards to the need for a cell lysis step. Certain AAV serotypes, such as AAV2, are poorly.

Disruptive technology and future innovation

Gene therapy manufacturing presents a unique opportunity to turn the treatment paradigm toward curative approaches. However, future innovation is key and there are several opportunities for disruptive and advanced manufacturing approaches.

Conclusion

Despite the increasing demand for AAV vectors, significant manufacturing challenges remain, affecting product cost and the ability to meet the needs of key therapeutic approaches. Current vector development has focused on improvements in vector design to improve vector targeting, with significantly less attention paid to the underlying factors affecting vector production, quality, stability and manufacturability. Going forward, there is an increasing need to increase the scale of production and reduce costs. The only way to achieve this is through standardization of the manufacturing process - and to make this feasible, the assessment of manufacturability needs to be a key criterion in the selection of candidate vectors.

References

Selvaraj, N.; et al. Detailed Protocol for the Novel and Scalable Viral Vector Upstream Process for AAV Gene Therapy Manufacturing. Hum Gene Ther. 2021 Aug;32(15-16):850-861.
Adams, B.; et al. Moving from the bench towards a large scale, industrial platform process for adeno-associated viral vector purification. Biotechnol Bioeng. 2020 Oct;117(10):3199-3211.
Hitchcock, T. Manufacturing of AAV vectors: translational challenges from development to industrialisation. Emerg Top Life Sci. 2021 Nov 12;5(5):725-728.
Selvaraj, N.; et al. Detailed Protocol for the Novel and Scalable Viral Vector Upstream Process for AAV Gene Therapy Manufacturing. Hum Gene Ther. 2021 Aug;32(15-16):850-861.

* For research use only. Not intended for any clinical use.

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The Key to Moving Gene Therapy from the Clinic to Commercialization Scaling Up the AAV Production Process

Challenges in scaling up AAV-based gene therapy manufacturing

Production process development: the cornerstone of AAV carrier scale up production

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