Background: Combinatorial complexity is a challenging problem in detailed and mechanistic mathematical modeling of signal transduction. This subject has been discussed intensively and a lot of progress has been made within the last few years. A software tool (BioNetGen) was developed which allows an automatic rule-based set-up of mechanistic model equations. In many cases these models can be reduced by an exact domain-oriented lumping technique. However, the resulting models can still consist of a very large number of differential equations.

2,4,6-Trinitrophenol (picric acid) and 2,4-dinitrophenol were readily biodegraded by the strain Nocardioides simplex FJ2-1A. Aerobic bacterial degradation of these -electron-deficient aromatic compounds is initiated by hydrogenation at the aromatic ring. A two-component enzyme system was identified which catalyzes hydride transfer to picric acid and 2,4-dinitrophenol. Enzymatic activity was dependent on NADPH and coenzyme F 420 . The latter could be replaced by an authentic preparation of coenzyme F 420 from Methanobacterium thermoautotrophicum. One of the protein components functions as a NADPH-dependent F 420 reductase. A second component is a hydride transferase which transfers hydride from reduced coenzyme F 420 to the aromatic system of the nitrophenols. The N-terminal sequence of the F 420 reductase showed high homology with an F 420 -dependent NADP reductase found in archaea. In contrast, no N-terminal similarity to any known protein was found for the hydride-transferring enzyme.

Protein aggregation of monoclonal antibodies (mAbs) is a common phenomenon associated with the production of these biopharmaceuticals. These aggregates can lead to adverse side effects in patients upon administration, thus expensive downstream processing steps to remove the higher molecular weight species are inevitable. A preferable approach is to reduce the level of aggregation during bioprocessing by a careful adjustment of critical process parameters. Recently, new analytical methods enabled characterization of mAb aggregation during bioprocessing of mammalian cells. Furthermore, rapid and efficient bioprocess optimization has been performed using design of experiments (DoE) strategies. In this work, we describe a DoE-based approach for the analysis of process parameters and cell culture additives influencing protein aggregation in Chinese hamster ovary (CHO) cell cultures. Important bioprocess variables influencing the aggregation of mAb and host cell proteins were identified in initial screening experiments. Response surface modeling was further applied in order to find optimal conditions for the reduction of protein aggregation during cell culture. It turned out that a temperature-shift to 31 °C, osmolality above 420 mOsm/kg, agitation at 100 rpm and 0.04% (w/v) antifoam significantly reduced the level of aggregates without substantial detrimental effects on cell culture performance in our model system. Finally, the aggregation reducing conditions were verified and applied to another production system using a different bioprocess medium and another CHO cell line producing another mAb. Our results show that protein aggregation can be controlled during cell culture and helps to improve bioprocessing of mAbs, by giving insights into the protein aggregation at its origin in mammalian cell culture.

Initial F420-dependent hydrogenation of 2,4,6-trinitrophenol (picric acid) generated the hydride sigma-complex of picrate and finally the dihydride complex. With 2,4-dinitrophenol the hydride sigma-complex of 2,4-dinitrophenol is generated. The hydride transferring enzyme system showed activity against several substituted 2,4-dinitrophenols but not with mononitrophenols. A Km-value of 0.06 mM of the hydride transfer for picrate as substrate was found. The pH optima of the NADPH-dependent F420 reductase and for the hydride transferase were 5.5 and 7.5, respectively. An enzymatic activity has been identified catalyzing the release of stoichometric amounts of 1 mol nitrite from 1 mol of the dihydride sigma-complex of picrate. This complex was synthesized by chemical reduction of picrate and characterized by 1H and 13C NMR spectroscopy. The hydride sigma-complex of 2,4-dinitrophenol has been identified as the denitration product. The nitrite-eliminating activity was enriched and clearly separated from the hydride transferring enzyme system by FPLC. 2,4-Dinitrophenol has been disproven as a metabolite of picrate (Ebert et al. 1999) and a convergent catabolic pathway for picrate and 2,4-dinitrophenol with the hydride sigma-complex of 2,4-dinitrophenol as the common intermediate has been demonstrated.

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