The impact of fluid-dynamic-generated stresses on chDNA and pDNA stability during alkaline cell lysis for gene therapy products.

Extensive tests have been carried out to assess the impact of fluid-dynamic-generated stress during alkaline lysis of Escherichia coli cells (host strain DH1 containing the plasmid pTX 0161) to produce a plasmid DNA (pDNA) solution for gene therapy. Both a concentric cylinder rheometer and two stirred reactors have been used, and both the alkaline addition and neutralization stages of lysis have been studied. Using a range of shear rates in the rheometer, stirrer speeds in the reactors, and different periods of exposure, their impact on chromosomal DNA (chDNA) and pDNA was assessed using agarose gel electrophoresis, a Qiagen Maxiprep with a polymerase chain reaction (PCR) assay, and a Qiagen Miniprep purification with a UV spectrophotometer. Comparison has been made with unstressed material subjected to similar holding times. These tests essentially show that under all these conditions, <2% chDNA was present in the pDNA solution, the pDNA itself was not fragmented, and a yield of 1 mg/g cell was obtained. These results, together with studies of rheological properties, have led to the design of a 60-L, stirred lysis reactor and the production of high-quality pDNA solution with <1% chDNA after further purification.     

Fed-batch bioconversion of indene to cis-indandiol

The bioconversion of indene to cis-(1S,2R) indandiol, a key intermediate in the synthesis of Merck’s HIV protease inhibitor, CRIXIVAN™ can be achieved during the growth of a Rhodococcus strain. In a previous study, we reported on the application of multi-parameter flow cytometry for the measurement of indene toxicity to the strain, and found that concentrations up to 0.25 g/l of indene (0.037 g indene/g dry cell weight) in batch bioconversions did not influence cell physiology. Using this information, this study reports on the implementation of a single phase indene fed-batch bioconversion. Cytoplasmic membrane (membrane) integrity and membrane polarisation of a large number of cells were measured during such bioconversions using multi-parameter flow cytometry and compared to a control in order to assess any toxic effects of indene feeding. The results indicate that indene supply at a rate of 0.1 g/l/h is feasible without any deleterious effects on cell physiology. The delay in indene metabolism was significantly shorter, with lower concentrations of by-product formation, when it was added to the culture in the stationary phase than when it was added at the beginning of the exponential phase of the fermentation. cis-Indandiol production rates could be enhanced from 20 mg/l/h, in a previously reported silicone oil two-liquid phase system, up to 200 mg/l/h by a combination of suitable indene feeding rates in the stationary phase and operating with a high biomass concentration to limit the effects of toxicity. In addition, the yield of cis-indandiol on indene (g/g) was higher at 0.48 in the single phase system compared to 0.20 in the two-liquid phase system. However, the final concentration of cis-indandiol was considerably lower, possibly as a result of higher dehydrogenase activity resulting in an increased transformation of cis-indandiol to 1-keto-2-hydroxy indan. This study has demonstrated that it is feasible to feed indene directly in the stationary phase of the bioconversion using high biomass concentrations to obtain enhanced cis-indandiol formation rates as well as yields based on indene utilisation compared to a two-phase silicone oil system.     

A common genetic system for functional studies of pitrilysin and related M16A proteases

Pitrilysin is a bacterial protease that is similar to the mammalian insulin-degrading enzyme, which is hypothesized to protect against the onset of Alzheimer's disease, and the yeast enzymes Axl1p and Ste23p, which are responsible for production of the a-factor mating pheromone in Saccharomyces cerevisiae. The lack of a phenotype associated with pitrilysin deficiency has hindered studies of this enzyme. Herein, we report that pitrilysin can be heterologously expressed in yeast such that it functionally substitutes for the shared roles of Axl1p and Ste23p in pheromone production, resulting in a readily observable phenotype. We have exploited this phenotype to conduct structure-function analyses of pitrilysin and report that residues within four sequence motifs that are highly conserved among M16A enzymes are essential for its activity. These motifs include the extended metalloprotease motif, a second motif that has been hypothesized to be important for the function of M16A enzymes, and two others not previously recognized as being important for pitrilysin function. We have also established that the two self-folding domains of pitrilysin are both required for its proteolytic activity. However, pitrilysin does not possess all the enzymatic properties of the yeast enzymes since it cannot substitute for the role of Axl1p in the repression of haploid invasive growth. These observations further support the utility of the yeast system for structure-function and comparative studies of M16A enzymes.     

The influence of impeller type in pilot scale xanthan fermentations

The rheological complexity of Xanthan fermentations presents an interesting problem from a mixing viewpoint, because the phenomena of poor bulk blending and low oxygen mass transfer rates inherent in highly viscous fermentations (and their consequences) can be systematically investigated, even at the pilot plant scale. This study in a 150 L fermentor compares the physical and biological performance of four pairs of impellers: a standard Rushton turbine, a large diameter Rushton turbine, a Prochem Maxflo T, and a Scaba 6SRGT. Accurate in-fermentor power measurements, essential for the comparison of impellers in relation to operating costs are also reported. It is demonstrated that the agitator performance in Xanthan fermentations is very specific and the choice of which impeller to use in bioreactors to obtain enhanced performance is dependant on the applied criterion. None of the criterion favored the use of the standard Rushton turbine, therefore suggesting that there are strong grounds for retrofitting these impellers with either large diameter impellers of similar design or with novel agitators. In addition, fluid dynamic modeling of cavern formation has clearly highlighted the importance of a well mixed and oxygenated region for providing the capacity for high microbial oxygen uptake rates which govern Xanthan productivity and quality. Copyright 1998 John Wiley & Sons, Inc.     

Agitator speed and dissolved oxygen effects in xanthan fermentations

Agitation speed affects both the extent of motion in Xanthan fermentation broths because of their rheological complexity and the rate of oxygen transfer. The combination of these two effects causes the dissolved oxygen concentration and its spatial uniformity also to change with agitator speed. Separating these complex interactions has been achieved in this study in the following way. First, the influence of agitation speeds of 500 and 1000 rpm has been investigated at a constant nonlimiting dissolved oxygen concentration of 20% of air saturation using gas blending. Under these controlled dissolved oxygen conditions, the results demonstrate that the biological performance of the culture was independent of agitation speed as long as broth homogeneity could be ensured. With the development of increasing rheological complexity lending to stagnant regions at Xanthan concentrations >20 g/L, it is shown that the superior bulk mixing achieved at 1000 rpm, compared with 500 rpm, leading to an increased proportion of the cells in the fermentor to be metabolically active and hence higher microbial oxygen uptake rates, was responsible for the enhanced performance. Second, the effects of varying dissolved oxygen are compared with a control in each case with an agitator speed of 1000 rpm to ensure full motion, but with a fixed, nonlimiting dissolved oxygen of 20% air saturation. The specific oxygen uptake rate of the culture in the exponential phase, determined using steady-state gas analysis data, was found to be independent of dissolved oxygen above 6% air saturation, whereas the specific growth rate of the culture was not influenced by dissolved oxygen, even at levels as low as 3%, although a decrease in Xanthan production rate could be measured. In the production phase, the critical oxygen level was determined to be 6% to 10%, so that, below this value, both specific Xanthan production rate as well as specific oxygen uptake rate decreased significantly. In addition, it is shown that the dynamic method of oxygen uptake determination is unsuitable even for moderately viscous Xanthan broths. Copyright 1998 John Wiley & Sons, Inc.     

A mathematical model for agitation-induced fragmentation of Penicillium chrysogenum

The morphology of filamentous organisms in submerged cultures varies between the pelleted and the dispersed forms depending on the strain of organism and the culture conditions. The dispersed form consists of branched and unbranched hyphae (freely dispersed form) and clumps (filamentous material in aggregates). In agitated systems, the choice of impeller geometry as well as the total power input determines the mechanical forces that might affect the morphology of filamentous species (e.g. by fragmentation) with simultaneous effects on their growth and productivity. To find out more about fragmentation of Penicillium chrysogenum caused by mechanical forces of different impeller types and agitation intensities, a population balance model has been developed. The projected area measured by image analysis was used to characterise the morphology (size) of the mycelia. In the model, the kinetics of mycelial fragmentation were expressed by a breakage rate constant K, which was assumed to be only dependent on the agitation conditions. The fragmentation rate was considered to follow a first order process in size (area) which was based on assumptions made for the mechanism of mycelial break-up, and work reported in the literature. Previously published mean and distributional data from off-line fragmentation experiments in ungassed vessels of sizes from 1.4 to 180?l were used to validate the model. For the first time a model has been found that is capable of fitting changes in mycelial morphology caused by mechanical forces generated by different impellers at various power inputs and scales. Besides the mean projected areas of the mycelia, the model allowed simulations of the projected area distributions, and changes in those distributions because of the agitation. At the small scale (1.4?l), the breakage rate constant K could be correlated well with either impeller tip speed or the “energy dissipation/circulation function”, which is based on mycelial circulation through the impeller region. The simpler but commonly used power input per unit tank volume did not correlate K adequately. The scale up data showed that only the “energy dissipation/circulation function” correlated mycelial fragmentation well. The dependence of K on biomass concentration, and its detailed dependence (if any) on the fermentation conditions at sampling, which might indicate likely breakage mechanisms, remain to be elucidated.     

The impact of fluid mechanical stress on <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> during continuous cultivation in an agitated bioreactor

The effect of mechanical stresses generated by an extreme agitation intensity or a high aeration rate on growth parameters and cell physiology were studied during continuous cultivation of the Gram-positive bacterium Corynebacterium glutamicum. It is concluded that variations in agitation, aeration rate, or dO₂ concentrations down to about 1% of saturation do not damage the bacterial cells or cause a significant change in physiological response, as measured by flow cytometry, even though the cell size was slightly reduced.     

Operational intensification by direct product sequestration from cell disruptates: application of a pellicular adsorbent in a mechanically integrated disruption-fluidised bed adsorption process

A novel prototype adsorbent, designed for intensified fluidised bed adsorption processes, was assembled by the emulsification coating of 4% (w/v) porous agarose upon a zirconia-silica solid core. The adsorbent, designated ZSA (particle density 1.75 g/ml, maximum pellicle depth 40 microm), was subjected to physical and biochemical comparison with the performance of two commercial adsorbents (Streamline and Macrosorb K4AX). Bed expansion qualities and hydrodynamic characteristics (N, D(axl) and B(o)) of ZSA demonstrated a marked robustness in the face of elevated velocities (up to 550 cm/h) and biomass loading (up to 30% (ww/v)) disrupted yeast cells. Cibracron Blue derivatives of the pellicular prototype (ZSA-CB), evaluated in the batch and fluidised bed recovery of glyceraldehyde 3-phosphate dehydrogenase (G3PDH) from unclarified yeast disruptates, exhibited superior capacities and adsorption/desorption performance to the commercial derivatives. These advanced physical and biochemical properties facilitated a demonstration of the direct, mechanical coupling of bead-milling and fluidised bed adsorption in a fully integrated process for the accelerated recovery of G3PDH from yeast. The generic application of such pellicular adsorbents and integrated processes to the recovery of labile, intracellular products is discussed.     

Studies related to the scale-up of high-cell-density E. coli fed-batch fermentations using multiparameter flow cytometry: effect of a changing microenvironment with respect to glucose and dissolved oxygen concentration

Multiparameter flow cytometric techniques developed in our laboratories have been used for the "at-line" study of fed-batch bacterial fermentations. These fermentations were done at two scales, production (20 m(3)) and bench (5 x 10(-3) m(3)). In addition, at the bench scale, experiments were undertaken where the difficulty of achieving good mixing (broth homogeneity), similar to that found at the production scale, was simulated by using a two-compartment model. Flow cytometric analysis of cells in broth samples, based on a dual-staining protocol, has revealed, for the first time, that a progressive change in cell physiological state generally occurs throughout the course of such fermentations. The technique has demonstrated that a changing microenvironment with respect to substrate concentration (glucose and dissolved oxygen tension [DOT]) has a profound effect on cell physiology and hence on viable biomass yield. The relatively poorly mixed conditions in the large-scale fermentor were found to lead to a low biomass yield, but, surprisingly, were associated with a high cell viability (with respect to cytoplasmic membrane permeability) throughout the fermentation. The small-scale fermentation that most clearly mimicked the large-scale heterogeneity (i.e., a region of high glucose concentration and low DOT analogous to a feed zone) gave similar results. On the other hand, the small-scale well-mixed fermentation gave the highest biomass yield, but again, surprisingly, the lowest cell viability. The scaled-down simulations with high DOT throughout and locally low or high glucose gave biomass and viabilities between. Reasons for these results are examined in terms of environmental stress associated with an ever-increasing glucose limitation in the well-mixed case. On the other hand, at the large scale, and to differing degrees in scale-down simulations, cells periodically encounter regions of relatively higher glucose concentration.     

Agitation induced mycelial fragmentation of Aspergillus oryzae and Penicillium chrysogenum

Given the impact of mycelial morphology on fermentation performance, it is important to understand the factors that influence it, including agitation-induced fragmentation. The successful application of the energy dissipation/circulation function (EDC) to correlate fragmentation of Penicillium chrysogenum with agitation intensity and with different impeller types [5] has already been demonstrated. The EDC function takes into account the specific energy dissipation rate in the impeller swept volume and the frequency of mycelial circulation through that volume. In order to explore whether the EDC function can be used more generally to correlate fragmentation of different filamentous species, the present study extended the concept to agitation-induced, off-line fragmentation of Aspergillus oryzae grown in chemostat culture. The work shows that at EDC values off-line greater than that in the chemostat, fragmentation with different impellers can be correlated with the EDC. For EDC values less than those used in the chemostat, fragmentation did not occur. The earlier results of Jüsten et al. [5] with Penicillium chrysogenum are also reconsidered and found to behave similarly.