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. © 2006 Wiley Periodicals, Inc.