The impact of process environment changes on process performance is one of the most crucial process safety issues when cultivating mammalian cells in a bioreactor. In contrast, directed shifting of process parameters can also be used as an optimization tool providing higher cell and product yields. Compared to other strategies that also aim on the regulation of cell growth and protein expression process parameter shifts can be easily performed without reagent addition or even genetic modification of the host cell line. However, a successful application of changing process conditions implies a profound understanding of the provoked physiological changes within the cells. In a systematic approach we varied the dissolved oxygen tension (DOT), pH, and temperature of CHO cultures in controlled bioreactors and investigated the impact on growth, productivity, metabolism, product quality and cell cycle distribution using a recombinant CHO cell line expressing the highly glycosylated fusion protein Epo-Fc. We found the reduction of cultivation temperature and the reduction of (external) pH to exert the most significant effects on process performance by mainly reducing cell growth and metabolism. With respect to the cell line used we identified a set of parameters capable of affecting cell proliferation in favor of an increased specific productivity and total product yield. The well directed alteration of the process environment has emerged as a tool adequate for further process optimization applying a biphasic cultivation strategy.

Upon stable cell line generation, chromosomal integration site of the vector DNA has a major impact on transgene expression. Here we apply an active gene environment, rather than specified genetic elements, in expression vectors used for random integration. We generated a set of Bacterial Artificial Chromosome (BAC) vectors with different open chromatin regions, promoters and gene regulatory elements and tested their impact on recombinant protein expression in CHO cells. We identified the Rosa26 BAC as the most efficient vector backbone showing a nine-fold increase in both polyclonal and clonal production of the human IgG-Fc. Clonal protein production was directly proportional to integrated vector copy numbers and remained stable during 10 weeks without selection pressure. Finally, we demonstrated the advantages of BAC-based vectors by producing two additional proteins, HIV-1 glycoprotein CN54gp140 and HIV-1 neutralizing PG9 antibody, in bioreactors and shake flasks reaching a production yield of 1 g/l.

Regulation of cell growth and protein expression potentially results in a sustainable enhancement of the volumetric productivity in a fermentation process. Following a biphasic cultivation strategy the process initially passes through a cell proliferation phase to generate a sufficiently high viable cell mass. In the subsequent production phase cells are maintained viable and productive without significant cell proliferation leading to increased viable cell days and product yields. In a previous work we have shown that the well directed alteration of the process environment based on process parameter shifting is a promising tool to regulate cell growth and protein expression. In continuation of this work we investigated process parameters which have been identified to affect cell proliferation in favor of an increased specific productivity and total product yield in a series of biphasic batch cultivation experiments. In most of these processes the integral of viable cells and the specific productivity were increased leading to a significant improvement of both final product concentration and volumetric productivity. In addition, combined parameter shifts (pH 6.90/30 degrees C and pH 6.90/33 degrees C) exerted a synergistic effect on product quality. The loss of product sialylation which occurred at reduced temperatures was prevented by simultaneously reducing the external pH. In conclusion, biphasic cultivation based on combined shifting of process parameters is a suitable tool for controlling cell proliferation and protein expression of mammalian cells in a batch bioreactor leading to enhanced volumetric productivities and therefore offers an enormous potential for bioprocess optimization.