Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture

There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serum-free media into ones that do not contain any components of animal origin, or ‘animal-free media’. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animal-sourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1~ 3×10⁶ viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.