(692d) Morphological and Life Cycle Changes Exhibited at the up-Scaling of a Saccharomyces Cerevisiae High-Cell Density Fed-Batch Biopharmaceutical Process

In a previous study (Fu et al, 2014), we observed a clear difference in yeast cell volumes (determined by wet cell weight (WCW)) grown at 10L laboratory scale versus 10,000L manufacturing scale. That study also included metabolite analysis which suggested hypoxia during scale up. We hypothesized that hypoxia weakens the yeast cell wall during the scale up, leading to changes in cell permeability, and/or cell mechanical resistance. Cells treated with the zymolyase showed a more sensitive cell wall later in the production process at manufacturing scale, although cell viability from samples obtained at both scales were similar. In the current study we further examine the relationship between relative volumes of yeast cells to cell viability, size, morphology, and life cycle at laboratory scale (10L) and manufacturing scale (10,000L). Vi-cell and flow-cam methodologies demonstrate a larger sub-population of cells with aspect ratio 0.5 and �6 micron-diameter at 10,000L scale, corresponding to more one- and two-budded mothers in the larger scale process. In addition, results from a cell cycle study using flow cytometry and DAPI fluorescence microscopy demonstrated multi-budded cells having increased chromosomal content with a long residence in the M phase without completing cytokinesis. In summary, these results collectively suggest a possible relationship between the level of cell wall compromise with cell volume and the presence of a major sub-population of larger budding cells reflecting a longer residence in M phase.