A recombinant Chinese hamster ovary (CHO) cell line was used to express a humanized antibody. Product quality analysis of this humanized antibody showed the presence of free thiol, due to impaired cysteine residues in the Fab region. Decreased potency of this thiol Fab made it critical to minimize the levels of free thiol. In an effort to do this, we evaluated the effect of copper sulfate addition to the cell culture production medium. As a component of the production medium, copper sulfate can act as an oxidizing agent, thereby facilitating disulfide bond formation. Four concentrations of copper sulfate were added at the beginning of 2-L benchtop production cultures of the recombinant CHO cell line: 0, 5, 50, and 100 mu M. We found that these copper sulfate additions had no effect on cell growth or antibody production. However, a slight dosedependent depression in culture viability was observed. Analysis of the purified antibody showed that either the 50 or 100 mu M copper sulfate additions reduced the level of free thiol by more than 10-fold.