R. Mutharasan

Strategies to extend longevity of hybridomas in culture and promote yield of monoclonal antibodies

SummaryCell viability was improved by supplemental feeding of amino acids and vitamins in batch culture of hybridoma cells. Cells could be maintained over a 10 day period following exponential growth at a constant viable cell concentration of 2.1×106 cells/ml. Concentrations of monoclonal antibody (MAb) reached 140 mg/l, a value of nearly four times that found in typical batch culture. Lactate formation appeared to occur only during active exponential growth and not during the stationary phase.

Alcohol production by Clostridium acetobutylicum induced by methyl viologen

SummaryControlled batch experiments performed withClostridium acetobutylicum show that methyl viologen induces solvent production at near neutral pH. At a pH of 6.8, significant ethanol production was observed in presence of methyl viologen. At pH 5, production of butanol and ethanol are favored at the expense of acetone.

NADH levels and solventogenesis in Clostridium acetobutylicum: new insights through culture fluorescence

SummaryThe metabolic relationship between the solventogenic state in Clostridium acetobutylicum and intracellular NADH levels was investigated using culture fluorescence as a technique for continuous monitoring of in vivo NADH levels. Continuous culture experiments showed that a transition from acidogenic to solventogenic state was accompanied by a decrease in culture fluorescence, which was interpreted as a decrease in NADH level. It appears that NADH/NAD+ turnover rates may be more significant than NADH levels in determining the metabolic state of the cell. This result provides important new information on regulation of the intracellular reduction state in Clostridium acetobutylicum. Culture fluorescence is shown to be a useful technique for non-invasive on-line monitoring of the metabolic state in continuous acetone-butanol fermentations.

Factors governing lactic acid formation in long term cultivation of hybridoma cells

SummaryEffect of glucose concentration and pH on lactic acid formation was investigated in batch cultures of a hybridoma cell line. Lactate formation increases with growth rate. High glucose concentration leads to extensive lactate formation only during growth phase and not during stationary phase. Lactate formation also may serve to regulate extracellular pH to pH 6.8, provided conditions are favorable to maintain viability and if sufficient nutrients are present in the medium.

Effect of various glucose/glutamine ratios on hybridoma growth, viability and monoclonal antibody formation

SummaryCultivation of a model hybridoma cell line in Dulbeccos modification of Eagles minimal essential medium (DMEM) at various glucose and glutamine levels showed that maxmium cell growth was obtained in a broad range of glucose/glutamine ratios of 0.6 to 6.0. A new parameter called the viability index is proposed to quantify cell viability. A strong correlation between viability index and monoclonal antibody (MAb) concentration was found at all glucose/glutamine ratios investigated.

On-line measurement of hybridoma growth by culture fluorescence

Fluorescence measurement of viable hybridoma cell cultures provides a convenient method for monitoring the progress of a batch culture. It is shown that cell concentration changes as low as 35,000 cells/ml during initial stages of growth can be measured reliably. This sensitivity, however, decreases to 2 × 106 cells/ml at cell concentration greater than 2 × 106 cells/ml. The culture fluorescence of hybridoma culture is a characteristic property of the cell and the medium used. Consequently, processes in which the medium composition and cell lines are invariant, a direct on-line estimate of viable cell count can be made using the method investigated in this paper.

Manipulation of End-Product Distribution in Strict Anaerobes

From a general perspective, metabolism of high energy substrates can be considered as a series of biological redox reactions. In aerobes, the electrons produced during catabolism find oxygen as the terminal acceptor. On the other hand, strict anaerobes are campelled to find other means for disposing of these electrons by forming reduced products such as hydrogen, methane, alcohols, and others. Recent reports have suggested that the shift in metabolism towards ethanol formation in thermophilic ethanol producers is strongly influenced by the regulation of electron flow.’” In fact, we earlier reported that the product distributions (ethanol and lactate) in the strict anaerobe Thermoanaerobacter ethanolicus were strongly influenced by the redox potential of the culture medium.’” In these studies, the culture redox potential was altered by adding suitable reducing agents to the growth medium. Zeikus and co-workers noted that the addition of electron acceptors (or oxidants) in Thermoanaerobium brockii resulted in increased acetate yield and a decreased formation of reduced end products, ethanol, lactate, and hydrogen! In this paper, we will examine the quantitative effects of reducing-agent addition on end-product distribution in both Thermoanaerobacter ethanolicus and Clostridium acetobutylicum growth in batch and continuous cultures.

Culture fluorescence characteristics and its metabolic significance in batch cultures of Clostridium acetobutylicum

SummaryQualitative characteristics of changes in culture fluorescence during batch growth ofClostridium acetobutylicum [ATCC 824] have been determined. A solventogenic phase culture shows higher changes in normalized fluorescence levels per unit change in cell concentration compared with an acidogenic phase culture. This suggests that during solvent formation intracellular NADH levels are higher than in the acidogenic phase and that NADH availability may influence the onset of solventogenesis.

Physical refining of steel melts by filtration

The removal of nonmetallic inclusions from steel melts prior to casting has significant merit. Laboratory prepared steel melts containing carefully prepared alumina inclusions have been successfully filtered at 1600 °C. Two distinct types of filters were used: (i) tabular alumina packed bed (0.2 to 0.5 cm nominal diameter) and (ii) extruded monolithic alumina (400 cells per square inch). The kinetics of the filtration process have been modeled, and inclusion removal efficiency of up to 96 pct has been achieved in laboratory melts. The results show that inclusion removal efficiency is a strong function of melt velocity in the range of 0.08 to 0.68 cm per second and is weakly dependent on filter length. The type of filter utilized affects inclusion removal efficiency significantly. The inclusion capture kinetics and the filtration characteristics of the filter media tested are discussed.

Effect of Furfural and Ethanol on the Growth and Energetics of Yeast under Microaerobic Conditions

Developpement dun modele pour la croissance et la production dethanol par des levures (Saccharomyces cerevisiae) dans un chemostat micro-aerobie en regime permanent