The production of adeno-associated viruses (AAVs) has become increasingly important over the past 10 years: AAV vector-based gene therapy is currently the only in vivo gene therapy approved in the USA and Europe.

With adenovirus now the viral vector of choice, along with a growing interest in gene therapies, there is an increasing need for cost-effective vector manufacturing technologies and viral vector production platforms for human gene therapy products.

These platforms must be able to cover all stages of the gene therapy production process, including development and storage. From cloning design to high-titer virus purification, from initial viral vector production to preclinical and large-scale production, reliability and adaptability are key. This is where our flexible and scalable single-use solutions come in.

Key aspects of adeno-associated virus production

There are various approaches that can be taken for the development of AAV vectors but, irrespective of the specific chosen production process, the steps will include plasmid development and production, cell expansion, plasmid transfection, viral vector production, purification, and fill and finish.

The preferred method to ensure a long-term and reliable supply of the viral vector, and, in turn, reliable gene therapy products, is to develop a stable producer cell line in vitro, which, if required, allows for scale-up from preclinical to large-scale production.

Given the potential safety and efficacy risks associated with pre-existing immunity to AAV vectors, antibody titers are included as inclusion or exclusion criteria for gene transfer therapy protocols, while neutralizing assays are used to detect neutralizing antibodies (nAbs) as well as other non-antibody neutralizing factors.

Development and production of plasmids

The first step in the workflow requires the development and production of large quantities of plasmid DNA, which will subsequently be transfected into host cells for AAV production. Following its expression in bacteria, plasmid DNA is harvested and purified from the fermentation culture.

Cell line growth and transfection – viral vector production commences

During the transfection process, DNA or RNA is artificially introduced to producer cell lines. This enables gene function and protein expression to be studied, by enhancing or inhibiting the expression of specific genes of interest. Conventional AAV vector production usually utilizes either insect cells or mammalian cells. When designing transfection experiments, the biological properties of the host cell must be taken into consideration, as some promoters may function differently in different cell types while other serotypes are not well suited to particular transfection technologies.

Current adenovirus expression systems aim to be helper-free, i.e., avoid the use of helper viruses, but instead include the plasmid pHelper. In seeking to produce the large quantities of rAAV necessary for clinical use, companies are moving away from production systems that use adherent HEK293 cells - the current choice because they supply additional proteins necessary for growth – to more scalable technologies using cell lines grown in suspension cell culture. In addition, the optimization of transient transfection protocols using cell lines grown in suspension facilitates efficiency through large-scale production processes. Another factor to be considered is the choice of bioreactor as well as biocontainers.

Harvesting and purification

In addition to the cell type, other factors such as the transfection method and the health and viability of a cell line must be considered if transfection is to be successful. Different AAV serotypes have different characteristics; AAV serotype 1, for instance, is deemed slightly more effective at transduction than AAV serotype 2. It should be noted, however, that all AAV vectors have a small packaging capacity, which limits their use for many larger genetic elements.

Popular methods used for AAV purification are either based on affinity chromatography or the use of a cesium chloride (CsCl) density gradient, combined with ultracentrifugation. Following two rounds of centrifugation, the purified virus is extracted in the final step of the AAV vector production process.

Before any viral vectors can be released for clinical use, safety tests are conducted to ensure the plasmid DNA titers are free from any process- or product-related impurities and that they are compliant with any good manufacturing practice (GMP) or regulatory requirements.

Aseptic Filling

As the final step of the process in viral vector production, it is imperative for the dispensing of vector into single-use bioprocess containers to be sterile. To prevent any human error that could lead to the contamination of an entire batch of gene therapy products, it is recommended that the entire virus production process is automated.

Being fully modular, the RoSS.FILL platform allows for maximum flexibility and scalability of both the filling and draining processes. It is possible to scale from preclinical to large-scale production and allow the filling of unlimited volumes per batch, all at a speed of up to 300L per hour.

As the components used throughout the entire filling process are fully disposable, this ensures the completely safe handling of viral vectors or indeed any other cell culture.

What next? Safe storage solutions for AAVs, by Single Use Support

Once the vector manufacturing process has been completed, the viral vectors must be transferred from the bioreactor to be stored and/or shipped in a protected manner. This includes freezing (and subsequent thawing) to preserve the AAV vectors for future use.

The fully automated freeze-thaw platforms developed by Single Use Support provide reliable end-to-end solutions for fully controlled freeze/thaw processes involving any human gene therapy product or other pharmaceutical substance. They allow for full scalability, all the way from clinical trial to commercial bulk production. In addition, our freeze-thaw platforms are compatible with any batch size and any single-use bags from all established manufacturers, allowing the greatest possible flexibility.

AAV production workflow – where do we come in?