The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice. Restimulation of Tregs after 8–12 days of culture with CD28 costimulation in the presence of rapamycin resulted in >1000-fold expansion of Tregs in <3 wk. Next, we determined whether other costimulatory pathways could augment the replicative potential of CD28-costimulated Tregs. We observed that while OX40 costimulation augmented the proliferative capacity of CD28-costimulated Tregs, Foxp3 expression and suppressive function were diminished. These studies indicate that the costimulatory requirements for expanding Tregs differ from those for T effector cells and, furthermore, they extend findings from mouse Tregs to demonstrate that human postthymic Tregs require CD28 costimulation to expand and maintain potent suppressive function in vivo.

Physiological state multiplicity was observed in continuous cultures of the hybridoma cell line ATCC CRL‐1606 cultivated in glutamine‐limited steady state chemostats. At the same dilution rate (0.04 h−1), two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations (7.36 × 105 and 1.36 × 106 cells/mL). Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the TCA cycle for energy generation. The low‐efficiency steady state was achieved after innoculation by growing the cells in a nutrient rich environment, first in batch mode followed by a stepwise increase of the dilution rate to its set point at 0.04 h−1. The high‐efficiency steady state was achieved by reducing the dilution rate to progressively lower values to 0.01 h−1 resulting in conditions of stricter nutrient limitation. The high energetic efficiency attained under such conditions was preserved upon increasing the chemostat dilution rate back to 0.04 h−1 with a higher nutrient consumption, resulting in approximate doubling of the steady state cell concentration. This metabolic adaptation is unlikely due to favorable genetic mutations and could be implemented for improving cell culture performance by inducing cellular metabolic shifts to more efficient flux distribution patterns. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 675–683, 1999.

Metabolic flux analysis is a useful tool for unraveling relationships between metabolism and cell function. Material balancing can be used to provide estimates of major metabolic pathway fluxes, provided all significant metabolite uptake and production rates are measured. Potential sources of metabolizable material in many serum‐free media formulations are low molecular weight digests of biological material such as yeast extracts and plant or animal tissue hydrolysates. These digests typically contain large amounts of peptides, which may be utilized as amino acids. This article demonstrates the need for accounting for amino acids liberated from peptides in order to accurately estimate pathway fluxes in Chinese hamster ovary cells grown in a complex (hydrolysate containing) medium. A simplified model of central carbon metabolism provides the framework for analyzing external metabolite measurements. Redundant measurements are included to ensure the consistency of data and assumed biochemistry by comparing redundant measurements with their predicted values from a minimum data set, and by expressing the degree of agreement using a statistical “consistency index.” The consistency index tests whether redundancies are satisfied within expected experimental error. For chemostat steady states of CHO cultures grown in a hydrolysate‐supplemented medium, consistent data were obtained only when amino acids liberated from peptides were taken into account. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 324–335, 1999.

Asparagine linked (N‐linked) glycosylation is an important modification of recombinant proteins, because the attached oligosaccharide chains can significantly alter protein properties. Potential glycosylation sites are not always occupied with oligosaccharide, and site occupancy can change with the culture environment. To investigate the relationship between metabolism and glycosylation site occupancy, we studied the glycosylation of recombinant human interferon‐γ (IFN‐γ) produced in continuous culture of Chinese hamster ovary cells. Intracellular nucleotide sugar levels and IFN‐γ glycosylation were measured at different steady states which were characterized by central carbon metabolic fluxes estimated by material balances and extracellular metabolite rate measurements. Although site occupancy varied over a rather narrow range, we found that differences correlated with the intracellular pool of UDP‐N‐acetylglu‐ cosamine + UDP‐N‐acetylgalactosamine (UDP‐GNAc). Measured nucleotide levels and estimates of central carbon metabolic fluxes point to UTP depletion as the cause of decreased UDP‐GNAc during glucose limitation. Glucose limited cells preferentially utilized available carbon for energy production, causing reduced nucleotide biosynthesis. Lower nucleoside triphosphate pools in turn led to lower nucleotide sugar pools and reduced glycosylation site occupancy. Subsequent experiments in batch and fed‐batch culture have confirmed that UDP‐sugar concentrations are correlated with UTP levels in the absence of glutamine limitation. Glutamine limitation appears to influence glycosylation by reducing amino sugar formation and hence UDP‐GNAc concentration. The influence of nucleotide sugars on site occupancy may only be important during periods of extreme starvation, since relatively large changes in nucleotide sugar pools led to only minor changes in glycosylation. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 62: 336–347, 1999.

Rapamycin was used as a medium additive to slow the progression of CRL 1606 hybridomas through the cell cycle, under the hypothesis that such a modulation might reduce cell death. Cell cycle distributions for CRL hybridomas in the G1 phase of the cell cycle ranged from 20% to 35% during batch, fed-batch, and continuous culture experiments, independent of culture time, dilution rate, growth rates, or death rates. Rapamycin, an mTOR signaling inhibitor, immunosuppressant, and G1-phase arresting agent, was identified and tested for efficacy in restraining cell cycle progression in CRL 1606 hybridoma cultures. However, in the presence of 100 nM rapamycin, the percentage of cells in the G1 phase of the cell cycle during fed-batch cultures was only increased from 28% to 31% in control cultures to 37% to 48% for those with rapamycin. Accordingly, rapamycin only slightly reduced culture growth rate. Instead, the use of rapamycin more notably kept viability higher than that of control cultures by delaying cell death for 48 h, thereby enabling viable proliferation to higher maximum viable cell densities. Furthermore, rapamycin enhanced specific monoclonal antibody production by up to 100% during high-viability growth. Thus, over the course of 6-day fed-batch cultivations, the beneficial effects of rapamycin on viable cell density and specific productivity resulted in an increase in final monoclonal antibody titer from 0.25 to 0.56 g/L (124%). As rapamycin is reported to influence a much broader range of cellular functions than cell cycle alone, these findings are more illustrative of the influence that signal transduction pathways related to mTOR can have on overall cell physiology and culture productivity.