Factor IX (FIX) plays an important role in the blood coagulation cascade. When Chinese hamster ovary (CHO) cells producing recombinant human FIX were cultivated using a serum-free medium (SFM) containing 1.12 mM of Ca2+ and 0.82 mM of Mg2+, a significant amount of active FIX (aFIX) was converted into undesirable activated FIX (FIXa) in the later phase of batch culture. In an effort to improve aFIX production from CHO cells, the effect of Ca2+ and Mg2+ concentrations in the culture medium on the activation of aFIX to FIXa was investigated using SFM with various concentrations of Ca2+ and Mg2+ in the range of 0-1.0 mM. The highest aFIX concentration of 1.36 IU/mL. was obtained at 1.0 mM Ca2+ and 1.0mM Mg2+. but the activation of aFIX to FIXa in the later phase of culture was rapid and significant. In contrast at 0.5 mM Ca2+ and 1.0 mM Mg2+, the aFIX concentration of 1.33 IU/mL was obtained and did not decrease significantly in the later phase of culture. Taken together, lowering Ca2+ concentration from 1.0 to 0.5 mM inhibits the activation of aFIX to FIXa in the later phase of culture, fortifying the robustness of downstream bioprocessing. (C) 2009 Elsevier B.V. All rights reserved.