With the recent approvals of the first virally delivered gene therapy and two CAR-T cell therapies, these revolutionary fields are advancing at an impressive pace. Growth in the number of cell and gene therapy companies and clinical trials is highlighted in the most recent report from the Alliance for Regenerative Medicine:
- Globally, companies active in gene and cellular therapies and other regenerative medicines raised more than $4.1 billion in the second quarter of 2018, a 164% increase from Q2 2017.
- There were 977 clinical trials underway worldwide at the close of the second quarter of 2018.
While poised to offer significant benefits to patients, gene and T-cell therapies may be the most complicated approaches to combat disease ever contemplated, and many development and manufacturing challenges remain.
Rapid growth in the industry has led to increased demand for viral vector production to support pre-clinical and clinical requirements for both in vivo and ex vivo approaches. In vivo gene therapy applications are those in which the viral vector is injected directly into the patient. In contrast, ex vivo therapies such as CAR-T require genetic modification of cells outside the body and transfer back to the patient. Early successes for lower-dose indications, such as for diseases of the eye, have paved the way for more ambitious efforts, including in vivo gene therapies for indications that require a higher dose of the vector, further increasing demand.
As a result, the industry is struggling to keep pace with the unprecedented spike in demand for bioprocess systems for vector production. Many companies, including Corning, are taking actions to expand manufacturing capacity and meet customers’ needs for viral vector production systems that can efficiently provide higher yields.
For these novel therapies to achieve sustained commercial and clinical success, optimized and scalable cell culture processes for vector production at the requisite quality are essential. Unfortunately, viral vector production is complex, and the scale-out of production is a significant technical barrier to widespread commercialization.
Adeno-associated vectors
Consider adeno-associated virus (AAV) vectors. These vectors are among the most frequently used vehicles for introducing genetic material into cells for in vivo applications. AAV vectors can deliver a large genetic payload and, depending on the serotype, can be used to infect a broad range of tissues. Additionally, the vector elicits a relatively mild immune response, can transduce both dividing and non-dividing cells, and can lead to long-term, robust transgene expression.
AAV vectors are typically manufactured via transfection of plasmid DNA into human embryonic kidney cells (HEK293). The overall process yields approximately 5x109 vector genome copies (GC) per cm2 of culture surface. To sustain ongoing trials and earlier stage discovery, it is estimated that production processes must achieve 1015-1016 genome copies per cm2 of culture surface. Higher yields and greater upstream (transfection and growth) and downstream (harvest and purification) process efficiencies are also needed to help reduce the cost of manufacturing.
Scale-out of adherent cells used for viral vector production requires expansion of the 2D surface area, preferably in an optimized spatial footprint. A number of solutions exist in this area including technology that provides an increase in the number of layers and corresponding cell growth surface area compared to traditional rigid single or multi-layered culture vessels. One cell culture solution uses a gas-permeable polystyrene film to provide gas exchange between cells and culture medium and the atmospheric environment, reducing head space and optimizing the growth area in the vessel. Individual modules of the vessel can provide 6,000 cm2 cell growth surface area and can be joined together to form a 36-layer vessel offering 18,000 cm2, representing 2.5 times more cell growth area per volumetric footprint compared to traditional cell culture vessels.
Image: Optimized and scalable cell culture processes for vector production are essential for gene therapy success. Image courtesy of Corning Life Science.
The advantage of such a large growth area in a single unit is that it enables single manipulations of large numbers of cells, reducing the risk of contamination that invariably increases with the requirement to handle hundreds, if not thousands of individual bottles. It also allows development of a “closed” system of tubing allowing easy seeding and harvesting of the chamber without exposing the chamber to the external environment. This is important in the production setting as these manipulations can then be performed outside a tissue culture hood or clean room.
In addition to increased surface area, some vessels utilize a specialized surface treatment to reduce the aromatic groups and increase the oxygen containing functional groups of the polystyrene backbone. This treatment increases the oxygen content of the polymer surface resulting in improved hydrophilicity and wettability, which improves cell spreading and attachment. In particular, this surface treatment has been shown to improve attachment of cells that may adhere poorly due to cell phenotype, stressful culture conditions (such as may occur in low serum conditions) or those that normally require additional biological coatings for attachment.
Anchorage and suspension cells
Another challenge in increasing capacity is the fact that viral vector production is typically achieved using anchorage-dependent cells, which are more difficult to scale than suspension cells. The field is exploring ways to adapt production systems to make use of suspension cultures. In addition to increasing capacity, efforts to meet yield requirements in a smaller footprint, make greater use of automated systems and conversion from planar surfaces to 3-dimensional cultures are expected to help address capacity requirements.
The recent approvals of AAV-mediated gene therapy Luxturna™ (Spark Therapeutics), Yescarta® (Gilead), and Kymriah® (Novartis) were watershed moments for the industry and for patients. We are at the beginning of what will be a remarkable era in medicine. As new gene and cell therapies rapidly progress from an idea at the lab bench to commercialization, manufacturing capabilities must keep pace. Developing new manufacturing strategies and optimizing every step of both upstream and downstream processes will help deliver on the promise of gene therapy. New approaches that ensure scalable, cost- and resource-efficient processes leading to safe and reproducible production will help enable this novel therapeutic modality to fulfill its promise.
Paul Spear, Ph.D., is bioprocess product line manager at Corning Inc. He can be reached at spearp@corning.com.