In perfusion mode, media and other nutrients are continuously exchanged and product is harvested throughout the culture period. Thus, the process operates as a chemostat with cell retention, where the constant addition of fresh medium and the elimination of waste products provides the cells with the stable environment they require. This enables much higher cell densities to be achieved and, in turn, leads to higher productivity.
Once the culture has reached a steady state, the culture may be maintained for up to 100 days. Since product is harvested as it is produced, the mean residence time of the product in the reactor is low and degradation is less likely. Indeed, daily output from a perfusion system run with perfusion rates of up to two reactor volumes per day can be equal or even greater than the output from an entire fed batch. Thus, perfusion technology is highly suited to products that show toxicity or other adverse effects toward host cells and to unstable products (e.g., sensitive to proteolytic degradation, desialyation, deamidation, or low solubility at production pH).
Capital and start-up costs are lower for perfusion technologies, as smaller upstream and downstream capacity is required, and the process uses smaller volumes and fewer seed steps than batch methods. Perfusion is also more amenable to development, scale-up, optimization, parameter sensitivity studies, and validation.
The cost of batch failure due to contamination is reduced in perfusion technologies. Should contamination occur, product harvested prior to contamination will not be affected and can still be processed. If infection arises early in the run, then only a small volume of media will have been used, and if it arises later, significant product will have been harvested. In either scenario, the cost of failures is reduced when compared with fed batch, where generally the whole run will be lost.
Despite the potential benefits of the perfusion mode, fed batch has been the most widely used production methodology in recent years. In part, this is due to fed-batch culture being well characterized and in part due to the expertise within the industry. This is beginning to change however and there are now several examples of commercial production using perfusion including factor VIII (ReFacto®) and IgG (Remicade® and Simulect®).
There are several reasons why perfusion technology is on the increase:
•Perfusion-control technology and general support equipment is improving, and the overall process is increasingly robust.
•The technology allows for scalability and bioreactors of a size up to a working volume of 1,000 L can be used, dependent on process.
•Cell-retention systems and the scalability of these, which have always been the achilles heel of this mode of operation, are improving, and that leads to less cell loss and cell death and greater viable cell densities than seen previously.
•Higher cell density is being achieved with greater than 5x107 cells/mL being regularly achieved compared to 2x107 in fed-batch cultures.
•All of the operation improvements have led to low infection rates and reliable results.
All of these factors lead to higher product concentration in the harvest and better yields without significant increase in cost. Typically, cost of goods in the 1,000 L bioreactor scale compares with fed-batch cost in the 10,000–20,000 L.