Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines

PI3Kinase signaling in glioblastoma

Glioblastoma (GBM) is the most common primary tumor of the CNS in the adult. It is characterized by exponential growth and diffuse invasiveness. Among many different genetic alterations in GBM, e.g., mutations of PTEN, EGFR, p16/p19 and p53 and their impact on aberrant signaling have been thoroughly characterized. A major barrier to develop a common therapeutic strategy is founded on the fact that each tumor has its individual genetic fingerprint. Nonetheless, the PI3K pathway may represent a common therapeutic target to most GBM due to its central position in the signaling cascade affecting proliferation, apoptosis and migration. The read-out of blocking PI3K alone or in combination with other cancer pathways should mainly focus, besides the cytostatic effect, on cell death induction since sublethal damage may induce selection of more malignant clones. Targeting more than one pathway instead of a single agent approach may be more promising to kill GBM cells

Enhancement of antibody production by the addition of Coenzyme-Q10

Recently, there has been a growing demand for therapeutic monoclonal antibodies (MAbs) on the global market. Because therapeutic MAbs are more expensive than low-molecular-weight drugs, there have been strong demands to lower their production costs. Therefore, efficient methods to minimize the cost of goods are currently active areas of research. We have screened several enhancers of specific MAb production rate (SPR) using a YB2/0 cell line and found that coenzyme-Q10 (CoQ10) is a promising enhancer candidate. CoQ10 is well known as a strong antioxidant in the respiratory chain and is used for healthcare and other applications. Because CoQ10 is negligibly water soluble, most studies are limited by low concentrations. We added CoQ10 to a culture medium as dispersed nanoparticles at several concentrations (Q-Media) and conducted a fed-batch culture. Although the Q-Media had no effect on cumulative viable cell density, it enhanced SPR by 29%. In addition, the Q-Media had no effect on the binding or cytotoxic activity of MAbs. Q-Media also enhanced SPR with CHO and NS0 cell lines by 30%. These observations suggest that CoQ10 serves as a powerful aid in the production of MAbs by enhancing SPR without changing the characteristics of cell growth, or adversely affecting the quality or biological activity of MAbs

Effects of hydroxyurea on monoclonal antibody production induced by anti-mIgG and LPS stimulation on murine B cell hybridomas

Chemical treatment with hydroxyurea (HU) has been selected as a simple and low cost strategy to generate a cell population enriched for the G1 phase. After the chemical treatment with HU, cells were stimulated with anti-mIgG to test if the positive effects of anti-mIgG on CD40 expression and specific IgG2a production rate were improved upon a cell population with a higher percentage of cells in G1 phase at the beginning of the cell culture. In addition, other treatments assayed in this work were the cell stimulation with Lipopolysaccharide (LPS) both before and after the HU treatment. It has been observed that the use of HU under conditions able to maintain the cells in viable state (0.1 mM for 20 h), has a negative effect on CD40 expression and specific IgG2a production rate induced by anti-mIgG. The positive effect of LPS on cell stimulation induced by anti-mIgG is reduced on cells treated with HU