Scale Up AAV Production for Gene Therapy | Corning

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.

Advanced in AAV Production

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 AAV 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.."

From Bench to Billions: Facilitating Scale-Up of Adherent Cultures for Cell and Gene Therapies

Corning Ascent Fixed Bed Reactor

The Ascent FBR system is a new linear-scalable platform for AAV production from Corning. Bongiorno explains "It is a fixed bed reactor that offers a huge amount of surface area in a small footprint. We currently offer from 1 m2 up to 5 m2 in the Ascent FBR system Process Development unit. We are developing pilot and production scale units that will use that same technology and go up to 1,000 m2 in a single unit. The Ascent system is a much more automated system than HYPERStack. It includes an integrated controller that automatically maintains pH, oxygen and temperature throughout the course of the run."

What makes the Ascent system unique compared to other bioreactors on the market is the geometry of the layers of mesh that provide an intensified surface area for cell growth. The Ascent FBR bioreactor’s novel design enables evenly distributed cell growth and subsequent uniform transfection efficiency and AAV production. According to Bongiorno, "We've designed it so we can get uniform fluid flow. If you have uniform fluid flow, you can get uniform attachment of your cells. Once you have that uniform attachment, you can also get uniform growth because you have uniform nutrient availability and waste removal. We've seen that the cells grow and produce very well in that kind of setup."

Start Strong

Once you have chosen a platform and an initial target scale, Bongiorno recommends first working with your cells on the new platform to confirm that everything is growing as expected. While HYPERFlask and HYPERStack vessels and many other Corning products use polystyrene for the growth surface, Ascent FBR uses PET. The surface treatment is the same, but testing that your cells can attach is advisable. In Bongiorno's experience, any necessary adjustments are most commonly related to the harvest procedure, such as the amount of time needed for protease treatment.

"It's really, really important with the Ascent FBR system and also with HYPERStack vessels to engage our technical team and get training," says Bongiorno. "With the Ascent FBR system, we offer a demo where our team will come onsite for multiple days and guide you through that first usage from start to finish, and we also offer hands-on training with HYPERStack manipulations. You do want to get your operations down and working the right way so that you can really optimize your cell seeding, expansion, transfection, and harvest processes."

As an example, researchers at the University of Pittsburgh successfully scaled up from HYPERStack vessels to the Ascent FBR system in their work on an AAV gene therapy for diabetes, allowing them to move from small animal studies to primate studies.

Learn more about why the future is exciting with the Ascent FBR system.