Adherent Cell Culture

Adherent cells are cells which must be attached to a surface to grow. They are commonly used in laboratory environments. However, to produce biopharmaceuticals, the preference has been to use suspension cells, often Chinese hamster ovary (CHO) cells, in traditional stirred tank reactors. Typically, most suspension cells were originally adherent and have been adapted to work in suspension culture. However, not all adherent cell lines can adapt to suspension culture in a swift. The change can influence the quality and yield of the product they are intended to produce. This applies certainly for virus producing cell lines, common for production of viral vaccines or gene therapies. Viral vaccines, cell therapies and gene therapies depend heavily on adherent cell culture but producing high enough quantities of adherent cells to meet capacity requirements has been a challenge. Large scale bioreactors have been designed and developed mainly to meet the challenges of growing suspension cell lines and to produce monoclonal antibodies (mAb). To grow adherent cell lines, manufacturers were for a long time relying on roller bottles or cell-trays. These are good laboratory-scale approaches, but have significant disadvantages when it comes to growing large quantities of adherent cells under cGMP (current Good Manufacturing Practices) conditions.

Industrializing virus production: cGMP grade virus manufacturing for viral vaccines and gene therapy

Early on in therapeutic development, viral vectors are manufactured using transient transfection in adherent cells on 2-dimensional systems such as cell factories or roller bottles. These processes are sufficient for Phase I/II trials which are typically only a few patients. Scale-up to Phase III/commercial-scale, requires a change in manufacturing to achieve the amount of virus required to treat even a small patient population. As gene therapy evolves, it will move towards indications requiring high amounts of virus either because of the tissue targeted (e.g. muscular dystrophy) or the large patient populations (e.g. hemophilia, cystic fibrosis).

Pall’s iCELLis® fixed-bed bioreactor is the only single-use bioreactor that has been specially designed to produce high quantities of cGMP grade virus for viral vaccine or gene therapy applications. This unique technology provides the cells with a 3-dimensional controlled growth environment with low shear stress, resulting in excellent cell growth and high virus productivity. It provides up to 500 m² of growth surface area, equivalent to 790 10-layer multi-tray stacks, in a small footprint. It has similar automation and control options compared to other single-use bioreactors, with integrated pH, dissolved oxygen (DO) and optional biomass sensors. This technology is currently being adopted by vaccine and gene therapy manufacturers worldwide, allowing them to break through the barriers of traditional cell culture methods and industrialize their therapies.

The white paper, "An end-to-end process for large-scale adenovirus manufacturing for gene therapy" describes the development of a robust integrated adenoviral manufacturing platform using adherent cells grown in the iCELLis fixed-bed bioreactor. The resulting well-controlled operational manufacturing process allowed the production of large amounts of high quality viral vector in compliance with cGMP requirements, suitable for the clinical trials.

Often, the barrier to further development of a therapy is not the quantity of cells needed, but the lack of automation and controls traditional 2D adherent cell culture systems have. For these applications, Pall has developed the Xpansion® multiplate bioreactor system that offers a 2-dimensional growth surface (up to 12 m²) in a closed and tightly-controlled environment. This bioreactor supports multiple applications but is mainly used for seed train operations for the iCELLis bioreactor, and to grow cGMP grade adherent stem cells for cell therapy.

For allogeneic stem cell therapies, which require large quantities of cells, the best approach can be the development of a microcarrier-based process, combined with a suitable single-use stirred tank bioreactor. In order to take a full advantage of the benefits of the technology, microcarriers can be delivered gamma-irradiated, ready-to-use, in a single-use system that connects directly to the bioreactor. In addition, harvesting the cells and separating from the microcarriers is required to proceed to further processing steps after the bioreactor. The Allegro® STR family of single-use bioreactors is an excellent bioreactor to run microcarrier processes, e.g. for human mesenchymal stem cell expansion.