Scientific Publications by FDA Staff

Biotechnol Prog 2012 Sep;28(5):1138-51

Bioreactor environment sensitive sentinel genes as novel metrics for cell culture scale-down comparability.

Kondragunta B, Joshi BH, Han J, Brorson KA, Puri RK, Moreira AR, Rao G

Scale-down of bioreactors is currently done based on matching one or more measurable parameters such as k(L) a, P/V, which could result in insufficient process comparability. Currently there is a lack of genomic translational studies in cell culture scale-down, which could help delineate measurable cellular attributes for improved scale-down. In this study, we scaled-down from a typical bench-scale 5 L bioreactor to a novel high-throughput 35 mL minibioreactor based on matching oxygen-transfer-rate, which resulted in cell growth and product related discrepancies using Sp2/0 cells. Performing DNA microarrays on time-course samples from both systems we identified approximately 200 differentially expressed transcripts, presumably due to bioreactor aeration and mixing differences during scale-down. Evaluating these transcripts for bioreactor-relevant cellular functions such as oxidative stress response, DNA damage response etc., we chose 18 sentinel genes based on their degree of difference and identified functionality, which we further verified by qRT-PCR. Tracking the differential expression of Sod1, Apex1, and Odc1 genes, we were able to correlate sparging-related damage and poor mixing, as possible causes for physiological changes such as prolonged culture in minibioreactors. Additionally, to verify our sentinel gene findings we performed follow-up improved scale-down studies guided by gene analysis, and measured transcriptomic changes. As a result, qRT-PCR based genomic profiles, and cell growth profiles showed better convergence between the improved minibioreactor conditions and the model 5 L bioreactor. Our results broadly show that based on the knowledge from transcriptomic changes of sentinel gene profiles, it is possible to improve bioreactor scale-down for more comparable processes.