Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production.

Glycoengineering enabled the production of proteins with human N-linked glycans by Pichia pastoris. This study used a glycoengineered P. pastoris strain which is capable of producing humanized glycoprotein with terminal galactose for monoclonal antibody production. A design of experiments approach was used to optimize the process parameters. Followed by further optimization of the specific methanol feed rate, induction duration, and the initial induction biomass, the resulting process yielded up to 1.6 g/L of monoclonal antibody. This process was also scaled-up to 1,200-L scale, and the process profiles, productivity, and product quality were comparable with 30-L scale. The successful scale-up demonstrated that this glycoengineered P. pastoris fermentation process is a robust and commercially viable process.

Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast.

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9gL(-1) vs. 1.2gL(-1)), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.

Metabolic and morphological differences between rapidly proliferating cancerous and normal breast epithelial cells.

The metabolic and morphological characteristics of two human epithelial breast cell populations--MCF7 cells, a cancerous cell line, and 48R human mammary epithelial cells (48R HMECs), a noncancerous, finite lifespan cell strain--were compared at identical growth rates. Both cell types were induced to grow rapidly in nutrient-rich media containing 13C-labeled glucose, and the isotopic enrichment of cellular metabolites was quantified to calculate metabolic fluxes in key pathways. Despite their similar growth rates, the cells exhibited distinctly different metabolic and morphological profiles. MCF7 cells have an 80% smaller exposed surface area and contain 26% less protein per cell than the 48R cells. Surprisingly, rapidly proliferating 48R cells exhibited a 225% higher per-cell glucose consumption rate, a 250% higher per-cell lactate production rate, and a nearly identical per-cell glutamine consumption rate relative to the cancer cell line. However, when fluxes were considered on the basis of exposed area, the cancer cells were observed to have higher glucose, lactate, and glutamine fluxes, demonstrating superior transport capabilities per unit area of cell membrane. MCF7 cells also consumed amino acids at rates much higher than are generally required for protein synthesis, whereas 48R cells generally did not. Pentose phosphate pathway activity was higher in MCF7 cells, and the flux of glutamine to glutamate was less reversible. Energy efficiency was significantly higher in MCF7 cells, as a result of a combination of their smaller size and greater reliance on the TCA cycle than the 48R cells. These observations support evolutionary models of cancer cell metabolism and suggest targets for metabolic drugs in metastatic breast cancers.

Establishment of higher passage PER.C6 cells for adenovirus manufacture.

PER.C6 cells, an industrially relevant cell line for adenovirus manufacture, were extensively passaged in serum-free suspension cell culture to better adapt them to process conditions. The changes in cell physiology that occurred during this passaging were characterized by investigating cell growth, cell size, metabolism, and cultivation of replication-deficient adenovirus. The changes in cell physiology occurred gradually as the population doubling level, the number of times the cell population had doubled, increased. Higher passage PER.C6 (HP PER.C6) proliferated at a specific growth rate of 0.043 h(-1), 2-fold faster than lower passage PER.C6, and were capable of proliferation from lower inoculation cell densities. HP PER.C6 cell volume was 16% greater, and cellular yields on glucose, lactate, oxygen, and amino acids were greater as well. In batch cultures, HP PER.C6 cells volumetrically produced 3-fold more adenovirus, confirmed with three different constructs. The increase in productivity was also seen on a cell-specific basis. Although HP PER.C6 were more sensitive to the "cell density effect", requiring lower infection cell densities for optimal specific productivity, they proliferated more after infection than lower passage PER.C6, increasing the number of cells available for virus production. The extensive passaging established HP PER.C6 cells with several desirable attributes for adenovirus manufacture.