The Chinese hamster ovary cell line (CHO cell) is considered by many as the workhorse of the biotechnology industry. It is a preferred cell for production of an ever-expanding array of engineered products that have therapeutic, diagnostic or other applications. The cell line not only provides a well characterized laboratory for performing genetic manipulations, but also offers an excellent expression host for production of engineered products. The glycosylation pattern of this mammalian cell is an advantage not offered by potentially more productive microbial expression systems. Inherent in its mammalian nature, however, is its relatively slow growth rate and the constraints on its cultivation.
Although great improvements have been made in the culture of most animal cells, resulting in increases in their productivity, their use in large scale production has been limited in the past to batch or fed-batch processes. In both processes, cells are inoculated into a fixed culture volume and the more product needed, the larger the culture system. Cell growth is rapid in the fresh medium. As the nutrients are consumed, and waste products accumulate, growth rate declines, resulting in a culture decay phase. In a fed-batch system, the viability and productivity of the culture can be extended somewhat by supplementing it with growth promoting additives. In either case, however, declining growth and decay is a consequence of consumption of essential nutrients and accumulation of growth limiting cellular byproducts. The final productivity of such cultures may typically vary from hundreds of mg/L to a few mg/L.
Alternatively, it is possible to extend and maintain the viability of a culture and its productive phase by continuously removing the inhibitory waste byproducts while replenishing the culture with fresh nutrient medium, in a process known as perfusion. Normally, this process is done using a filter or other cell retention device, where the protein product is collected with the waste stream while the cells remain in the reactor. Continuous removal of product through the waste stream prevents its accumulation in the vessel. Rapid removal and storage offers an important advantage with some labile products, but generally, the large volume collected is not desirable. The ATF System used in a concentrated fed-batch (CFB) process takes advantage of the high cell concentrations and resultant high levels of product formation offered by perfusion and at the same time eliminates dilution of the product. The ATF System CFB process is realized simply by using a standard molecular weight cut-off filter, typically, 50 kDa. The filter allows waste products or any compound smaller than the indicated pore size to flow through, but not larger molecules. Antibodies and many other potential proteins or products larger than 50 kDa are retained by the filter. Both cells and product can thus be concentrated to high specific concentrations. A comparison between a standard fed-batch and CFB was undertaken by Biovian to evaluate the differences between these two processes.MATERIALS AND METHODS
ATF System: Refine Technology Alternating Tangential Flow (ATF2) System using the F2 Hollow Fiber Module 50 KDa pore, PS, 1 mm ID, 0.14 m2
Wave reactor: Wave Cellbase 20 PS with control unit Wave Biotech BWC
Wave bag: Sartorius-Stedim Cultibag RM 2 L Optical ATF and Opta SFT sterile connector
Medium: HyClone SFM4CHO
CHO cells: CHO cell line expressing human immunoglobulin G (hIgG)
Analysis of product: hIgG immunoassay.
The CHO cell seed culture was prepared in spinner flask. The cells were transferred to the wave reactor, at a starting concentration of approximately 0.4x106 cells/mL (Day 1). The bag culture-volume was approximately 1 L. The bag was equipped with optical sensors for dissolved oxygen (DO) and pH on-line measurements. Aeration was not initiated until Day 2 in order to maintain pH. Afterwards, aeration was maintained at 100 mL/min. The oxygen concentration was maintained as instructed by the manufacturer by increasing the rock and angle parameters.
In the fed-batch reference run, nutrients, such as CD-feed and glucose, were fed batch-wise at four different points in time during the process. The pH was automatically controlled.
In the ATF CFB experiments, the ATF System was connected to the culture bag. The flow was started on Day 2 at a constant flow rate of 0.5 L/min. The perfusion (fresh medium in and waste medium out) was started on Day 3. In Run 1, the perfusion rate was initiated at 0.8 L/d and increased to 1.2 L/d. In Run 2, it was initiated at 0.8 L/d and increased to 2.5 L/d by Day 7. The medium addition and waste outflow rates were determined by weight change of the respective containers.
Samples of approximately 2 mL were removed at least once a day from the cultivation bag with a syringe. DO and pH were registered during the sampling. An aliquot of the sample was immediately frozen at –20 °C for product analysis performed after the end of the actual experiment. Glucose and lactate concentrations were determined subsequent to sampling. The cell concentration and cell viability were analyzed by diluting the sample in phosphate buffered saline and tryphan blue before automatic counting of living and dead cells by a cell counter. The product was analyzed by a hIgG immunoassay.