Adeno-associated virus (AAV) vectors are commonly used for biological research and have enormous potential for treating human diseases through gene therapy. As preliminary research leads to pre-clinical and clinical research, the process of scaling up cell culture production to a manufacturing scale has historically been a major bottleneck. A common approach has been to adapt cell lines for suspension culture so that they can be grown in up to 1000-liter or larger stirred tank bioreactors, which is far from ideal, since most research is conducted with adherent cells.
Recent advances have greatly simplified the process of scaling up production of adherent cells and reduced the hands-on time, space, and cost associated with each unit of adeno-associated virus. Here we will walk through the gene therapy scale-up process, from small scale to large scale, with insight from Tom Bongiorno, PhD, Field Application Scientist for Corning Life Sciences. Bongiorno explains, "Once you induce cells to produce virus, they essentially stop proliferating, so you need to expand your producer cell line up to the total number of cells needed, start your transfection and then collect your virus."
Corning® HYPER technology vessels and Corning Ascent® Fixed Bed Bioreactor (FBR) can facilitate the scale-up process. The best choice depends on the scale of production needed in the short and long term.
Corning HYPER Technology
The Corning HYPERFlask® is similar to a traditional 175 cm2 T-flask in size and shape, but has 10 layers of ultra-thin polystyrene for 1720 cm2 of growth area. According to Bongiorno, "In the HYPER system, gas exchange is actually occurring through that gas-permeable polystyrene film, rather than in headspace. Depending on the cell type, we often see that the cells actually get better gas exchange in HYPER than they would in a traditional flask, and they grow a bit faster and produce a bit better as a result."
The next size up from the HYPERFlask is the Corning HYPERStack® vessel, which uses the same geometry and gas-permeable polystyrene film, but is a closed system. HYPERStack vessels are available with 12 or 36 layers (6,000 and 18,000 cm2, respectively) and can be manifolded together to increase yield, reduce handling time, and decrease the risk of contamination. Bongiorno finds that "HYPERStack is good for customers who may be transitioning from a more traditional 2D scale and want to continue that same type of process. It is going to be relatively labor-intensive compared to something like the Ascent FBR system, but you will have fewer demands in terms of expertise. Customers who are familiar with the process in a Petri dish can transition to HYPERStack with less additional training than an advanced instrument like Ascent.."