Tetanus toxin production in soy-based medium: nutritional studies and scale-up into small fermentors.

AIMS: To further improve the soy-based medium, devoid of animal and dairy products, for a production of tetanus toxin by nutritional studies and to scale-up the Clostridium tetani process into small fermentors. METHODS AND RESULTS: Optimum production of tetanus toxin did not require addition of pantothenic acid, thiamine, riboflavin, pyridoxine, biotin and uracil, growth factors used by previous investigators. Furthermore, l-tyrosine and l-cysteine could be eliminated from our soy-based medium without effect. Seven carbon sources were compared with glucose in the soy-based medium, but none was found to be superior to glucose. The process was successfully scaled-up into 250-ml bottles, 1-l bottles and 1-l fermentors. CONCLUSIONS: Quite remarkably, when comparing the tetanus production process in our soy-based medium with the traditional animal/dairy-containing media, our medium does not require addition of expensive vitamins, uracil or carbon sources other than glucose. Furthermore, the l-tyrosine and l-cysteine components could be eliminated, making the medium (Hy-Soy, glucose, powdered iron and inorganic salts) much more simple and economical. The successful scale-up from test tubes into 1-l fermentors allows us to predict that further scale-up into large fermentors will be successful. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (BSE; mad cow's disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. Our vegetable-based process avoids such unfortunate possibilities. The medium, having been made simpler and less expensive, and shown to be scaleable from test tubes into small fermentors, should be excellent for large scale production of tetanus toxin.

Menstrum for culture preservation and medium for seed preparation in a tetanus toxin production process containing no animal or dairy products.

AIMS: To completely eliminate animal and dairy products from the lyophilization menstrum and the seed medium used to produce tetanus toxin with Clostridium tetani. METHODS AND RESULTS: Tetanus toxin production in a recently developed fermentation medium lacking animal and dairy products was studied with different seed media. It was found that soy peptone could completely replace the beef heart infusion plus animal peptone previously used as seed medium. In addition, we found that cells lyophilized in soy milk could replace the usual type of cells lyophilized in cow's milk. CONCLUSIONS: We have now developed a complete tetanus toxin production process containing no animal and dairy products. SIGNIFICANCE AND IMPACT OF THE STUDY: Toxoid preparations made from toxin produced with animal and dairy products can contain undesirable contaminants such as the prion causing bovine spongiform encephalopathy (Mad Cow's Disease) or antigenic peptides that stimulate anaphylactic reactions and other undesirable immune reactions in immunized hosts. The new vegetable-based process described here avoids such unfortunate possibilities.

Quantitative measurements of mixing intensity in shake-flasks and stirred tank reactors. Use of the Mixmeter, a mixing process analyzer.

A new mixing probe has been developed which measures the motions of the fluid during mixing as pressure fluctuations and converts the measurements into a mixing signal (MS). The MS is the root mean square (RMS) pressure fluctuation in the 1-64Hz range as determined by a sensitive pressure sensor and a digital signal processor specifically designed for the purpose. The MS is a measure of the actual mixing flow of the fluid rather than a measurement of the input motions or energies into the reactor system (e.g. RPM, torque or power). In other studies, the MS has been measured as a function of mixing speed in numerous sized reactors from 10 to 1000l, and provides consistent and reproducible measurements. The MS increases monotonically as a function of mixing speed, with a change of slope corresponding to the transition from laminar to turbulent mixing regimes. Maps of MS as a function of location in the reactor are useful in understanding stirred tank reactor design and performance. Quantitative measurements of mixing are especially useful during process development as a tool to increase the success of scale-up during the transition from process development to manufacturing. Measurements at a fixed location in a given reactor are useful in understanding changes in mixing that occur during the course of a given process, and are useful in manufacturing situations where validated documentation of lot-to-lot consistency of mixing is required (e.g. pharmaceutical manufacturing). In addition, the probe has been used to measure mixing in vessels with vibrational mixers with similar results. The probe has been successfully used in feedback loops to control either mixing speed or vibrational mixing amplitude in order to maintain constant mixing of the fluid during processing. With this system it is possible to maintain constant mixing over a wide range of fluid volumes in a given reactor, and, for instance, to compensate for changes in viscosity throughout the course of the process. Adaptations of this system for the measurement of mixing in shake-flasks is described in this paper.

Development of the optimal inoculation conditions for microcarrier cultures.

The environmental conditions under which anchorage-dependent mammalian cells are grown are not necessarily those under which a culture should be initiated. Cell attachment is a physical process, and those factors which affect forces involved in cell attachment differ from the biological factors which affect cell growth. We have conducted an extensive experimental study to define clearly the optimal environmental conditions for MRC-5 cell attachment onto microcarriers. These inoculation conditions are particularly important when the serial propagation of mammalian cells on microcarriers is considered as in a human vaccine production process. The conditions which were investigated are: initial serum content (% v/v), initial pH, inoculation level (cells/bead), agitation rate (rpm), and the concentration of microcarriers (g/L). The initial distribution of attached cells was found to have a significant affect on the overall efficiency of anchorage-dependent cell cultures, and was used to evaluate attachment efficiency. Based on the experimental results, we propose an optimized protocol for the inoculation of microcarrier cultures.

Cell surface energy and membrane associated actin in lymphocytes.

We have shown previously that membrane associated actin correlates with the migratory abilities of lymphocytes during recirculation, and that cell surface energy correlates with the adhesiveness of lymphocytes to other cells. In this study, measurements of actin content and cell surface energy have been made for various lymphocyte subpopulations to examine the possibility that recirculation ability may be related to nonspecific adhesiveness. We have found that: both cell surface energy and actin content combine to provide a consistent explanation for the relative rates of recirculation of various lymphocyte subpopulations, and cell surface energies and actin contents vary independently in these lymphocyte subpopulations. Comparison of the actin contents and cell surface energies of metastatic and nonmetastatic lymphoma cell lines indicated that the differences in metastatic potential were more likely attributable to specific receptor-ligand interactions than to nonspecific adhesiveness. Cell surface energy and actin content are consistent with the greater adhesiveness of B cells than T cells to nylon wool, providing a physical basis for this common cell separation technique.

Optimal control of substrate concentrations in bioreactors with separated sensors.

Many of the sophisticated sensors desirable for monitoring bioreactors cannot be placed in the bioreactor either because they are not steam sterilizable or because they require nonphysiological operating conditions. Such sensors can be used if they are separated from the bioreactor. Separation of the sensor from the bioreactor causes a time lag in data acquisition. This results in several complexities in the development of an appropriate and stable feedback control system based on a separated sensor. This paper analyzes the optimal control of a bioreactor with a separated sensor without a time lag and analyzes the feedback control (but not necessarily the optimal control) with a time lag. Simulation results indicate that this type of analysis could be extended to more general bioreactor operating conditions.

Control of nitrate concentration in fermentations of Corynebacterium glutamicum.

A nitrate control system has been devised for the maintenance of stable nitrate concentrations throughout fed-batch fermentations of Corynebacterium glutamicum. The feedback control system was based on the use of a nitrate-ion-selective electrode to directly monitor the nitrate levels in the fermentor and an automatic controller to activate a nitrate feed pump. The electrode which was used for controlling the nitrate level was stable through-out the fermentation period. The apparent maximum specific growth rate, biomass production, protein production, biomass yields on glucose and nitrate, and amino acid production were all optimal at approximately 50mM nitrate.

Control of ammonium concentration in Escherichia coli fermentations.

A control system has been devised for the maintenance of stable ammonium concentrations throughout a fedbatch fermentation. The control system is based on an ammonia gas-sensing electrode that monitors a pH-adjusted effluent stream from the fermentor. To overcome the time lag between the fermentor and the electrode, feedback control included metered flows of ammonium to both the fermentor and the electrode vessel. The system was used to study the growth of Escherichia coli B (ATCC 11303) at controlled ammonium concentrations of 5 to 200mM. Apparent specific growth rates, biomass and protein production, and glucose yields were essentially constant from 5 to 170mM. Above 170mM ammonium growth was inhibited. As ammonium concentration decreased from 170 to 5mM, ammonium yields increased from 1 to 24 g cell dry wt/g ammonium utilized. The results demonstrate that control of ammonium concentrations at levels so low that ammonium would be exhausted in batch fermentations can significantly increase overall ammonium yields.

Interference of cyclosporin with lymphocyte activation maintenance of the cells in a non-cycling phase and with a low intracellular pH.

Cyclosporin can block T cell activation by mitogens as evidenced by inhibition of exogenous thymidine integration into DNA and by blockage of the cells in the G0/G1 phase of the cell cycle. The mitogenic activation sequence shows at least one cyclosporin sensitive step at or prior to the rise of cytoplasmic pH (pHi), which normally occurs after exposure to mitogen and which seems to be a permissive condition for initiation of DNA synthesis (S phase of the cell cycle).

Intracellular pH and the cell cycle of mitogen-stimulated murine lymphocytes.

Rapidly proliferating, polyclonally stimulated mouse spleen lymphocytes were separated by density-gradient unit-gravity sedimentation. The following measurements were made on each fraction: the average intracellular water volume, the distribution of DNA content by flow microfluorometry, the rate of 3H-thymidine incorporation, and the intracellular pH. Fractions of cells with a small average intracellular volume were predominantly in G0 or G1 phase of the cell cycle, while fractions of larger cells had higher proportions of cells in S or G2. Multiple regression analysis of the data for both T and B lymphocytes indicated that the intracellular pH of cells in G0, G1, or G2 is around pH 7.2, and that the intracellular pH of cells in S phase of the cell cycle is around pH 7.4.

Adhesion, phagocytosis and cell surface energy. The binding of fixed human erythrocytes to rat macrophages and polymethylpentene.

Fixed human erythrocytes were used as model particles for the study of adhesion and phagocytosis by rat peritoneal macrophages. Erythrocytes were fixed with various concentrations of glutaraldehyde or tannic acid, or were treated with neuraminidase. Adhesion and phagocytosis of these cells were measured. In addition, the surface energy of these erythrocytes and macrophages was estimated by the contact angle technique. Free energies of adhesion, based on the cell surface energies, were correlated with both adhesion and phagocytosis.

High intracellular pH accompanies mitotic activity in murine lymphocytes.

Intracellular pH is crucial to the control of many cellular processes such as glycolysis, protein synthesis, and cell division. To study the relation between intracellular pH and mitotic activity in actively dividing Con A- or LPS-stimulated splenic lymphocytes, a method was developed to determine intracellular pH using a fluorescence-activated cell sorter. The new method uses the pH-sensitive fluorochrome, 4-methylumbelliferone. Results obtained with it not only correspond qualitatively with the results obtained using 14C-dimethyloxazolidinedione (DMO) but also clearly show the active and inactive subpopulations. The intracellular pH of mitogen-stimulated murine lymphocytes increases from pH 7.15 to pH 7.45 when the population has greatest mitotic activity. The intracellular pH of three virus-transformed lymphocyte cell lines is higher by approximately 0.5 pH units when the cells are in exponential growth compared to stationary phase.

Intracellular pH of mitogen-stimulated lymphocytes.

Mitogenic stimulation of mouse lymphocytes results in two sequential intracellular alkalinizations. The first shift of intracellular pH from 7.18 to 7.35 coincides with early biochemical events following mitogenic stimulation. The second alkalinization begins 12 hours after stimulation and rises in parallel with the rate of thymidine incorporation. The results suggest that intracellular alkalinization following stimulation may play a key role in the enhancement of cellular activation and mitogenesis.

Determination of cell/medium interfacial tensions from contact angles in aqueous polymer systems.

The contact angles on cell layers of a series of polymeric droplets from aqueous two-phase systems of dextran and poly(ethylene glycol) have been used to determine the critical or limiting interfacial tension for spreading on the cell layers. Test droplets of the denser dextran-rich phase were formed in the lighter poly(ethylene glycol)-rich phase. The interfacial tensions, gamma, between the phases were determined with the pendant drop method, and a linear relationship was found between gamma-1/2 and the cosine of the angle the droplets made with the cell layers (Good-Girifalco plot). We were thus able to determine the limiting or critical interfacial tension, gamma c, for spreading on the cell layers. The value of gamma c is a measure of the interfacial energy of the cell/bathing medium interface. Values of gamma c obtained by this method include the following: 0.65 and 0.84 microN . m-1 for human erythrocytes and neutrophils, respectively; 0.93 microN . m-1 for porcine pulmonary macrophages; 0.75--3.60 microM . m-1 for various transformed murine lymphoid cell lines, and 2.53 microN . m-1 for Balb/c murine spleen lymphocytes. Exposure to various agents has differing effects on gamma c. Concanavalin A reduces gamma c, and bacterial lipopolysaccharide increases gamma c of murine spleen lymphocytes. The calcium ionophore, A23187, increases gamma c of both porcine pulmonary macrophages and murine spleen lymphocytes. This new method provides a quantitative approach to the cell surface energy and hydrophobicity which are thought to play an important role in membrane-mediated phenomena and in cell adhesion.

The effect of substrate on inhibition of Corynebacterium lepus by isonicotinic acid hydrazide (Isoniazid).

Isonicotinic acid hydrazide (INH) inhibits the growth of Corynebacterium lepus on hexadecane but has no effect on its growth on fructose. INH also inhibits the production of the mycolic acid containing lipopeptide bioemulsifier normally produced by C. lepus in response to an insoluble substate. The primary effect of INH appears to be inhibition of mycolic acid synthesis, which limits the growth of C. lepus on hexadecane by reducing the concentration of bioemulsifier.