Gene expression profiling for mechanistic understanding of cellular aggregation in mammalian cell perfusion cultures.

Aggregation of baby hamster kidney (BHK) cells cultivated in perfusion mode for manufacturing recombinant proteins was characterized. The potential impact of cultivation time on cell aggregation for an aggregating culture (cell line A) was studied by comparing expression profiles of 84 genes in the extracellular adhesion molecules (ECM) pathway by qRT-PCR from 9 and 25 day shake flask samples and 80 and 94 day bioreactor samples. Significant up-regulation of THBS2 (4.4- to 6.9-fold) was seen in both the 25 day shake flask and 80 and 94 day bioreactor samples compared to the 9 day shake flask while NCAM1 was down-regulated 5.1- to 8.9-fold in the 80 and 94 day bioreactor samples. Subsequent comparisons were made between cell line A and a non-aggregating culture (cell line B). A 65 day perfusion bioreactor sample from cell line B served as the control for 80 and 94 day samples from four different perfusion bioreactors for cell line A. Of the 84 genes in the ECM pathway, four (COL1A1, COL4A1, THBS2, and VCAN) were consistently up-regulated in cell line A while two (NCAM1 and THBS1) were consistently down-regulated. The magnitudes of differential gene expression were much higher when cell lines were compared (4.1- to 44.6-fold) than when early and late cell line B samples were compared (4.4- to 6.9-fold) indicating greater variability between aggregating and non-aggregating cell lines. Based on the differential gene expression results, two mechanistic models were proposed for aggregation of BHK cells in perfusion cultures.