Genetic studies indicated that the Drosophila melanogaster protein REAPER (RPR) controls apoptosis during embryo development. Induction of RPR expression in Drosophila Schneider cells rapidly stimulated apoptosis. RPR-mediated apoptosis was blocked by N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk), which suggests that an interleukin-1 beta converting enzyme (ICE)-like protease is required for RPR function. RPR-induced apoptosis was associated with increased ceramide production that was also blocked by Z-VAD-fmk, which suggests that ceramide generation requires an ICE-like protease as well. Thus, the intracellular RPR protein uses cell death signaling pathways similar to those used by the vertebrate transmembrane receptors Fas (CD95) and tumor necrosis factor receptor type 1.

Capacitance is used to monitor "biomass" in cell culture and fermentation processes. The present work explores various ways to support good manufacturing practices (GMP) use and create robust methods that can assure proper performance of capacitance equipment. An at-line capacitance measurement method is particularly valuable as it can be used to correct or confirm online measurements from bioreactors. To obtain consistent predictions of online capacitance, the at-line method requires appropriate control of culture temperature, vessel geometry, mixing, and timing. The at-line method yields values that differ systematically from online values, but the variation is ≤11% for two cell lines tested. A cell line-specific conversion factor addresses the difference and enables accurate predictions of online capacitance values. In addition to the at-line method, verification of equipment performance is further enabled by using formaldehyde-treated cell standards. The cell-derived standards create a stabilized system for studying capacitance and recapitulate the performance of metabolically active cell culture. The formaldehyde-treated cells, however, require a much greater conversion factor to predict online outputs. The stabilized system proves useful for evaluation and calibration of capacitance measuring systems. The at-line method and stabilized cell-derived suspensions support use of capacitance for process control in large scale mammalian cell culture.

Mammalian cells were grown to high density in a 3,000 L culture using perfusion with hollow fibers operated in a tangential flow filtration mode. The high-density culture was used to inoculate the production stage of a biomanufacturing process. At constant permeate flux operation, increased transmembrane pressures (TMPs) were observed on the final day of the manufacturing batches. Small scale studies suggested that the filters were not irreversibly fouled, but rather exposed to membrane concentration polarization that could be relieved by tangential sweeping of the hollow fibers. Studies were undertaken to analyze parameters that influence the hydrodynamic profile within hollow fibers; including filter area, cell density, recirculation flow rate, and permeate flow rate. Results indicated that permeate flow rate had the greatest influence on modulating TMP. Further evaluation showed a significant decrease in TMP when permeate flow was reduced, and this occurred without any negative effect on cell growth or viability. Hence, a 30% reduction of permeate flow rate was implemented at manufacturing scale. A stable operation was achieved as TMP was successfully reduced by 75% while preserving all critical factors for performance in the perfusion bioreactor.