Based on a Plackett-Burman (P-B) experimental design, a chemically-defined and protein-free medium, named Super-CHO protein-free (SPF), was developed to support an autocrine Super-CHO cell line, C2.8-SPF, to grow as a single-cell. suspension culture with a growth rate and viable cell number profile comparable to that observed in a commercial medium containing undefined additives. The C2.8-SPF was subsequently transfected with a metal-inducible and amplifiable expression construct, pNK-EH1, designed to produce a humanised anti-CD48 monoclonal antibody, IgG1-EH1 (EH1 MAb). This study demonstrated that while the statistical experimental design provided a simple method for medium optimisation of the host cell, the resulting formulation might not be optimal for derived cell lines and further modification (in this case, addition of iron, to ameliorate heavy metal toxicity) was required. Developments of Super-CHO C2.8-SPF autocrine cell line and protein-free medium (SPF) described here provide a low-cost and regulatory-compliant recombinant protein expression platform, suitable for the manufacture of therapeutic recombinant proteins. (c) 2007 Society of Chemical Industry.