ScaleUp and Adaptation to Suspension Serum Free Growth

Clones identified based on high specific productivity or mRNA expression are generally expanded on tissue culture plates until enough cells are available to inoculate them into a suspension culture. Depending on the cell type, two T-175 tissues culture flasks usually contain sufficient cells to inoculate a spinner flask with a 200 mL working volume with ~3-4 x 105 cells/mL. For cell lines that are maintained as adherent cultures in serum-containing media, suspension adaptation can often be accomplished quite readily in the same serum-supplemented media in spinner vessels maintained at 50-80 rpm. Altering the media or adding components that discourage adhesion of cells to solid substrates, or use of specially treated glass and plastic-ware can facilitate this process. The primary consideration in methods choice should be robustness and compatibility of the resulting condition of the cells to growth under manufacturing conditions. During this procedure it is critical to initiate or "seed" the cultures at a suitably high cell density and to monitor growth and viability closely over the first several passages. For cell lines that are resistant to culture in suspension, media optimization altering the concentrations of cations may be necessary (90). Several commercial vendors have specialized media available for this purpose.

Cell lines that are intended for commercial cell culture processes should also be adapted to serum-free or protein-free growth conditions early in development. In fact, the desire to eliminate as many animal-derived raw materials as possible from commercial cell culture operations has lead to the development in some instances of transfection and selection procedures which completely remove the need for serum in the process. Where this is not possible, adaptation of cells to serum-free growth is routinely performed. This process is not well understood physiologically and is technically more challenging than adaptation of cells to suspension growth in serum-containing media. Appropriate supplementation of the basal media powder with growth factors, trace elements, lipids and vitamins is critical for success (90,91). Some protocols call for slowly reducing the serum concentration of media, whereas others favor a complete removal of serum in a single step. In either case, cells often will show a slowed growth profile and decreased viability for several passages after the complete withdrawal of serum and need to be carefully monitored to ensure the success of this procedure. In many instances, several media exchanges using centri-fugation of cells away from spent media and resuspension in fresh media need to be performed in order to remove waste products and maintain an adequate seeding density (3-10 x 105 cells/mL). It is important that selective pressure be maintained on the gene of interest throughout this process to ensure retention of high-level gene expression. However, the concentration of selective agent and of supplemental growth factors may need to be temporarily modified during this procedure to ensure success.

After cells have adapted to suspension and/or serum-free growth, their growth properties are much closer to what they will be in a large-scale cell culture process.

At this stage the cell lines can be compared for volumetric productivity and product quality using assays designed to model conditions encountered in the extended, high-density culture conditions typical of large-scale industrial cell culture processes. Such assays usually involve both plate-based and small-scale bioreactor formats. Plate based designs provide the ability to look at a broader number of cell lines to develop a sense of which lines perform best under defined process conditions. However, performance of cells in these assays does not always mirror cell performance under the pH and dissolved oxygen-controlled environment of the bioreactor. Bioreactors allow information to be developed on a limited number of cell lines and for key process parameters that might alter a given cell lines performance to begin to be identified. Several new high throughput systems that more closely approximate the gas exchange and control typical of fermentation systems, without the complexity of fer-mentor set-up and monitoring offer an attractive compromise for cell line selection studies.

Many aspects of product quality, ranging from gross estimates of molecular weight and presence or absence of glycans to detailed analysis of glycan structure or charge variants using mass spectrophotometric and chromatographic techniques can be monitored during this final selection period to ensure that the final clone selected for further process development has the most desirable cell culture and product properties.

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