NS0 cells require exogenous cholesterol for growth. The non-glutamine synthetase (GS) cholesterol-dependent NS0 host was treated with 5-azacytidine (5azaC), a demethylation drug, and adapted to grow in cholesterol-free, chemically defined medium. Within 7 weeks, a stable, cholesterol-independent NS0 host (NS0.CF) was obtained. The new NS0.CF host, as well as the original cholesterol auxotroph host, was transfected with the same mAb expression plasmid, and the top producing clone from both hosts were compared side-by-side in the enhanced platform fed-batch cultures using chemically defined media. The NS0.CF derived clone significantly out-performed the cholesterol-dependent clone, with titer reaching 4.5?g/L versus 3.0?g/L, respectively, mainly due to higher specific productivity, while key product quality attributes remained comparable. This work demonstrated an effective and rapid approach to generate a cholesterol-independent NS0 host, and its application in recombinant protein production. Biotechnol. Bioeng. 2012; 109:16851692. (C) 2012 Wiley Periodicals, Inc.