Adeno-associated virus (AAV) has emerged as the leading vector for transferring genes in the treatment of various diseases. The AAV manufacturers such as CBM make sure of the safety profile and efficient transduction. However, large-scale production and long-term storage result in lower yields and reduced shelf-life. The manufacture of AAV viral vectors is a complicated process and requires creative approaches and careful execution according to clinical and market demand.
The viral vector manufacturing workflow has the following steps:
- Upstream process
- Downstream process
- Fill and finish
Various challenges are faced at each of these steps. Let’s have a look!
1. Plasmid Development
Plasmid DNA is mostly obtained from the fermentation of E.coli bacteria. In AAV manufacturing, the consistency of fermentation, yield variability and the final product are the major concerns. Producing a plasmid with 95% purity and minimal manufacturing variabilities is a major challenge. To cope with the rising demand for AAV, plasmid manufacturing needs to be improved.
2. Cell Expansion
HEK293-based transfection process is used to produce viral vectors on a small scale. But in larger production units, the process produces a lot of variabilities. The use of adherent cells in the scale-up process has many challenges like the risk of contamination, and difficulty in monitoring and regulating specific culture conditions. The use of animal-based products in cell expansion is the source of adventitious materials that act as contaminants; therefore there is an absolute need to minimize the use of animal-based products and to find new and better sources.
3. Cell Lysis
The cell lysis in the downstream process is generally carried out by repeated freeze/thaw followed by low-speed centrifugation. This process is not suitable when it comes to large-scale purification due to the difficulty in scaling up the process. Another method is the French Press which is scalable but it causes a huge product loss due to the movement of the cells through an orifice by high pressure. Hence, an alternate cell lysis method is required which can be scaled up and protects the cell from stress damage.
The most expensive unit in AAV manufacturing is AAV. Filtration can cause aggregation of the particles and loss of their functionality due to sheer stress in the process. A significant amount of cell debris is formed during the lysis process which is difficult to pass through filters. Optimizing a filter specifically for AAV manufacturers is a huge challenge because it also depends on the AAV serotypes being processed. Large holdups and product loss during filtration is a challenge which needs attention.
5. Separation of Empty capsids from Full Capsids
Empty capsids are either empty or contain a few segments of genetic material; hence, they are considered contaminations. The presence of empty capsids affects the safety and efficacy of the AAV vectors as their presence induces immunogenicity in the product. Separating empty capsids from full ones is a challenging process due to the similarity of size and charge. Anion exchange and caesium chloride chromatography are used to separate the capsids. Anion exchange causes capsid damage and the caesium chloride method is not scalable. Significant efforts are required to overcome this challenge.