Specific TNFR-Fc productivity (q(TNFR-Fc)) in CHO cells was found to decrease by 68% at a cell density of 6 x 10(6) cells/mL compared to that at 2 x 10(6) cells/mL. In order to unravel the reasons for the reduced q(TNFR-Fc) at high cell density, an in-depth study of physiological factors, gene transcription and post-translational process was carried out. Physiological factors including cell biomass, intracellular protein and cell cycle distribution were not affected by cell density. Transcriptional analysis revealed that TNER-Fc mRNA level at 2 x 10(6) cells/mL was almost 2-fold higher than that at 6 x 10(6) cells/mL. Moreover, rather than mRNA stability, the slower transcriptional rate led to the decreased mRNA level. By analyzing the post-translational process, we found that the protein assembly rate and transport rate from the endoplasmic reticulum to the Golgi at high cell density were significantly lower than those at low cell density. Therefore, the decreased q(TNFR-Fc) at high cell density was correlated with the reduction in both mRNA level and post-translational processing rate. It was further found that the decreased mRNA level and post-translational processing rate might be linked to the apparent increase in CO(2) partial pressure (pCO(2)) at high cell density, which had resulted in the reduction in intracellular pH in CHO cells. (C) 2011 Elsevier Ltd. All rights reserved.