(494e) Modulating Shear with a Population Balance Equation Model to Sustain Aggregate Size Distributions in Plant Cell Culture

One of the key characteristics of plant cell suspension cultures is the propensity for cellular aggregation due to the incomplete separation of cells during division. Aggregates can range in size from 30-2,000 µm, resulting in the formation of subpopulations within a culture. Subpopulations are believed to be a major cause of culture heterogeneity due to diffusion limitations within an aggregate, as well as differences in cell exposure to shear. Aggregation dynamics directly affect the production of high value secondary metabolites. In Taxus plant cell suspension cultures that are used commercially to supply the anti-cancer compound paclitaxel smaller aggregates have been shown to accumulate higher levels of paclitaxel. As a result, controlling aggregation through application of shear could lead to higher producing, more homogeneous cultures. Through mechanical shearing of Taxus suspension cultures, culture aggregation can be reduced without affecting culture health and growth. By applying constant levels of shear throughout the growth period, the mean aggregate size of a culture can be reduced by up to 200 µm over an unsheared flask. Despite the reduction in culture aggregation over 16 generations of cell growth, the mean culture diameter remained unstable, fluctuating by ~ 300 µm (between 180 and 480 µm) for the sheared population and by ~ 400 µm (between 256 and 645 µm) for the unsheared population.

To gain better control of the aggregation properties through modulation of shear, the key parameters that affect disaggregation of Taxus cultures in response to shearing were investigated. Results showed that the starting aggregate size distribution had a major effect on disaggregation, but the culture density, day of culture and cell line had no effect. This information was used to develop a population balance equation model for the control of aggregate size in Taxus cultures through shearing. Two adjustable parameters were estimated using non-linear optimization. To determine the amount of shear necessary to reduce aggregation of a culture to a desired aggregation profile, a target aggregate size distribution was utilized. The model was applied to determine shearing conditions necessary to maintain a culture at a constant mean aggregate size over multiple generations of cell growth. Development of a method to maintain constant aggregation properties in a plant cell culture system has the potential to reduce production variability and increase product yields. Additionally, by creating cultures with a smaller, more controlled mean aggregate size, the effect of aggregation on secondary metabolite production can be more systematically evaluated in both plants and other cellular systems.