Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.