A scale-down model must be statistically similar to its corresponding large-scale process. In this article from BioProcess International magazine, authors from Genzyme report on using 12-L bench-top bioreactors to model a 2,000-L microcarrier-based perfusion cell culture process. They scaled down agitation rate based on a power/working-volume ratio and matched scale-independent parameters to the commercial-scale process. A two one-sided test (TOST) helped them establish statistical equivalency to qualify the scale-down model. Comparing critical product-quality attributes showed that cell culture performance of the model was within specified ranges.
Materials and Methods
Cell Culture: We propagated Chinese hamster ovary (CHO) cells that express a recombinant therapeutic enzyme on macroporous microcarriers in 15-L stirred-tank Broadley James bioreactors with a 12-L working volume. These cultures operated in perfusion mode for about 65 days. After a short period of growth in batch mode immediately following inoculation, the system fed medium continuously into the bioreactors to provide nutrients, with harvest fluid removed using a proprietary cell separation device to collect the product of interest and to reduce metabolic waste accumulation (19). The cell-separation device uses gravity to separate microcarriers (with cells attached) from supernatant.
We used two different proprietary Genzyme media sequentially to provide nutrients (e.g., amino acids and vitamins) to the cultures. One medium type was used for the culture’s five day growth phase during which cells reached a targeted density for production. We used a second medium type for the remainder of the culture while harvesting product from the bioreactors (about 60 days). Product was harvested continuously into collection vessels before downstream processing. Parameters such as temperature, pH, and dissolved oxygen (DO) were monitored and controlled to a target set-point.
Culture pH was maintained within a desired operating range using pH probes (Broadley James) that were calibrated against a daily offline pH measurement using a Bayer Rapidlab 248 blood gas analyzer as needed. Dissolved oxygen probes (Broadley James) were used to maintain the DO set point at 50%, and the resulting oxygen sparge rate served as a secondary indicator for cell growth. Additionally, we used pCO2 probes (YSI Life Sciences) to monitor the cultures and target the pCO2 range at large scale. A DeltaV automation system (Emerson Process Management) with feedback control capability monitored and controlled pH, DO, temperature, and gases. We set up perfusion control (continuous feed and harvest) through the DeltaV system. All these parameters were matched to the large scale, with the exception of bioreactor vessel, impeller size, cell-separation device size, and harvest-collection vessel size.