Optimal NS0 cell growth in a hollow fiber bioreactor requires increased serum concentration or a cholesterol supplement on the cell side of the fiber.

An NSO/GS cell line secreting a humanized antibody was routinely propagated in a T-flask using 2% serum. For scale-up of antibody production, this cell line was inoculated into a hollow fiber system using the same serum concentration. The metabolic activity increased for a few days in the hollow fiber system, but invariably the activity dropped dramatically as the cells died by day 7. A hollow fiber micro-bioreactor was used as a screening tool to examine possible reasons for cell death in the large-scale system. As seen in the hollow fiber system, cells died when 2% serum was used either on the cell side only or on both sides of the fiber in the micro-bioreactor. In contrast, the use of 20% serum on the cell side of the fiber and basal medium on the non-cell side resulted in good cell expansion at high viability. Regardless of the cell side serum concentration, no further growth enhancements were seen when up to 20% serum was placed on the non-cell side of the fiber. These results suggest that a serum component that does not readily cross the fiber is limiting cell growth in the hollow fiber bioreactors. The addition of a cholesterol-rich lipid supplement resulted in better cell growth in the micro-bioreactor, while the addition of other non-cholesterol lipid supplements resulted in no growth enhancement. The growth-enhancing properties of the cholesterol supplement were more pronounced at lower serum concentrations, suggesting that poor growth at low serum concentration was due to suboptimal cholesterol levels. When the cell side serum concentration was increased to 20% in the hollow fiber system, cells grew and filled the bioreactor, allowing a 39-day production run. These results demonstrate that this NSO cell line requires an increased cell side serum concentration for optimal growth and that this requirement is likely due to the inherent cholesterol dependency of this cell line.

Use of hollow fiber systems for rapid and direct scale up of antibody production from hybridoma cell lines cultured in CL-1000 flasks using BD Cell MAb medium.

The combination of BD Cell MAb medium with the CL-1000 flask is increasingly being used to generate a few hundred milligram of antibody for early stage research projects. Cells are inoculated at 2 million per ml, and the antibody is harvested after 15 days or when the antibody concentration reaches above 10 mg ml(-1), whichever comes first. Currently, there is no means to scale up beyond this production level using this technology. In this study, we evaluated hollow fiber technology as the scale up alternative. The hollow fiber system was run in batch mode to mimic the method used for the CL-1000 with BD MAb medium. The FL-NS murine hybridoma cell line was simultaneously inoculated at 2 million cells per ml in a CL-1000 and the Maximizer hollow fiber bioreactor system, a 21-fold theoretical scale up over the CL-1000. The Maximizer produced 23-fold more antibody, very close to the expected theoretical amount. However, production was complete after 9 days in the Maximizer, while the CL-1000 required the full 15 days for production. In summary, these results demonstrate successful scale up of antibody production from the CL-1000 to a hollow fiber system.