Showing papers by "Paul F. Greenfield published in 1992"
TL;DR: A superior estimator based on the maximum likelihood principle was developed and discussed in relation to the choice between the endpoint titration assay and the plaque assay, and an alternative two-stage assay is presented.
Abstract: The statistics of estimators used with the endpoint assay for virus titration were investigated. For a standard assay with 10 wells/dilution, the graphical estimator traditionally used was found to produce estimates with significant positive bias and a relatively low accuracy. Furthermore, the graphical estimator was found to be inconsistent. A superior estimator based on the maximum likelihood principle was developed. The results are discussed in relation to the choice between the endpoint titration assay and the plaque assay, and an alternative two-stage assay is presented.
47 citations
TL;DR: It is concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear, and the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient‐rich basal media, in addition to the considerations of gas bubble effects described by other workers.
Abstract: Metabolic effects of the medium supplements, fetal bovine serum (FBS), Pluronic F68, and bovine serum albumin (BSA) were compared for agitated bioreactor cultures of hybridoma cells. Agitation speeds up to 600 rpm, without entrainment of gas bubbles by sparging or vortex formation, allowed examination of cell interactions with turbulent fluid forces. For cultures in FBS-supplemented RPMI media, there was no significant effect of intense turbulent fluid shear on cell growth, metabolism, or antibody, production. Serum-free cultures (Pluronic F68 or BSA supplements) at 600 rpm demonstrated greatly increased glycolysis rates during exponential growth relative to controls. Nutrient limitations caused increased rates of decline of the viable cell concentrations and a reduction in final antibody titers by around 70%. The Pluronic F68 and BSA supplements did not lead to cell protection by modifying metabolism under conditions of intense turbulent fluid shear. Supplementing the protein-free medium with FBS reduced glycolysis rates in exponential growth phase, but this did not prevent a high rate of viable cell decline and low antibody titers. We concluded that FBS does not have a metabolic effect on cells subjected to intense turbulent fluid shear. Although the agitation conditions employed in this study were more intense than generally required for agitated bioreactor culture of hybridomas, we have demonstrated the importance of considering metabolic effects of turbulent fluid forces on cultures using nutrient-rich basal media, in addition to the considerations of gas bubble effects described by other workers.
36 citations
TL;DR: The model is useful in aiding the design and optimization of large-scale systems for production of biological insecticides as well as recombinant proteins and in delineating those areas which are limiting the process and require further, more fundamental, investigation.
Abstract: A mathematical model has been developed to describe the growth and infection of insect cells by recombinant baculoviruses. The model parameters were determined from a series of independent experiments involving batch suspension culture. The profiles generated by the model for cell growth, virus production and protein production agree with those observed in experiments. Presently, the model simulates only systems where cells are not growth-limited. The model is useful in aiding the design and optimization of large-scale systems for production of biological insecticides as well as recombinant proteins and in delineating those areas which are limiting the process and require further, more fundamental, investigation.
33 citations
TL;DR: A standing column packed bed bioreactor was constructed to enable packed bed operation during solid-state cultivation (SSC) of sago starch by R. oligosporus UQM 145F to be followed by in situ drying of the microbial product.
Abstract: A standing column packed bed bioreactor was constructed to enable packed bed operation during solid-state cultivation (SSC) of sago starch byR. oligosporus UQM 145F, to be followed byin situ drying of the microbial product. The essential amino acid composition of the microbial product was determined from the samples obtained from the cultivation with a substrate loading of 250g.
13 citations
TL;DR: An unstructured model is developed to describe the growth and product formation behavior of a recombinant yeast Saccharomyces cerevisiae using β‐galactosidase as a model protein, which shows good agreement with the experimental data over a range of conditions.
Abstract: An unstructured model is developed to describe the growth and product formation behavior of a recombinant yeast Saccharomyces cerevisiae using beta-galactosidase as a model protein. The model shows good agreement with the experimental data over a range of conditions. It also accurately predicts the effect of growth rate on yield coefficient and gene expression. The simplicity and accuracy of the model make it suitable for designing and implementing control and optimization strategies for the production of recombinant proteins at large scale.
12 citations
01 Jan 1992
TL;DR: The production of a model recombinant protein (β -galactosidase) by rAcNPV infected Sf-9 (Spodoptera frugiperda) insect cells was enhanced by modification of the medium environment, indicating that the decrease in productivity that normally occurs at a high cell density of infection was totally reversible.
Abstract: The production of a model recombinant protein (β -galactosidase) by rAcNPV infected Sf-9 (Spodoptera frugiperda) insect cells was enhanced by modification of the medium environment. Improved production by chemical enhancer dimethyl sulphoxide (DMSO) occurred in both serum containing and serum free cultures. Addition of 1% DMSO immediately prior to infection or incorporated as a permanent medium component enhanced product yield by 100% without decreasing cell viability or density. Electron microscopy of DMSO treated insect cells revealed intracellular changes concurrent with enhanced recombinant protein production. Improved production was also achieved by medium exchange; which indicated that the decrease in productivity that normally occurrs at a high cell density of infection was totally reversible. The effect did not appear to be directly cell mediated but rather an effect associated with changes in the medium environment. Original medium was replaced by fresh medium immediately prior to infection which resulted in constant specific productivity of β -galactosidase at times of infection up to the period when maximum cell densities were achieved late in the stationary phase.
7 citations
TL;DR: Evidence of similar effects occurring during the normal span of continuous cultures fed enriched medium at low dilution rates (0.002–0.005 1/h) is presented and the effect of this observation on optimisation is discussed.
Abstract: Inhibition caused by rapid changes in the environment has earlier been observed in hybridoma cultures following deliberate step-changes in the culture environment. This paper presents evidence of similar effects occurring during the normal span of continuous cultures fed enriched medium at low dilution rates (0.002–0.005 1/h). The effect of this observation on optimisation is discussed. In continuous culture at a dilution rate of 0.013 1/h, a viable cell density of 4×109 cells/l was achieved by gradually increasing the nutrient concentration in the feed medium. The MAb titre was 200 mg/l representing a 6-fold increase compared to batch culture and a 2-fold increase compared to continuous culture using standard medium.
7 citations
01 Jan 1992
TL;DR: It was shown that not only growth rate but also antibody (MAb) production rate was changed under different DOTs, and results indicate that MAb titers can be increased 2–3 fold, through this approach.
Abstract: It has been observed that hybridomas are metabolically affected by environmental conditions, particularly during the growth stage. Dissolved oxygen tension (DOT) is considered an important engineering parameter for the cultivation of hybridomas, as oxygen limitations occur when culture volumes are increased, and due to the low solubility of oxygen in medium. In this work, it was shown that not only growth rate but also antibody (MAb) production rate was changed under different DOTs. To optimise the growth rate and MAb production rate, strategies of controlling DOT were developed to improve MAb production levels. Different fermentation modes with varying DOT profiles were operated and the results indicate that MAb titers can be increased 2–3 fold, through this approach.
1 citations
TL;DR: An enriched serum free medium, SF900 II, and enhancer developed by Gibco for improving recombinant protein production by the Baculovirus Expression System using Sf9 cells has been evaluated and had a slight effect on production of s-gal.
Abstract: An enriched serum free medium, SF900 II, and enhancer developed by Gibco for improving recombinant protein production by the Baculovirus Expression System using Sf9 cells has been evaluated Production using SF900 II matches that shown by the commonly used TNMFH 10% FCS medium, but has additional benefits for downstream purification given that it is serum free and essentially protein free The enhancer had a slight effect on production of s-gal particularly in terms of production per cell Proteolysis of product is potentially a serious problem when serum free media is used
1 citations
01 Jan 1992
TL;DR: A relatively simple, unstirred, non-aerated tank is the most common bioreactor in industrial use today and many of the processes taking place in such reactors are anaerobic and used to generate wine, potable and industrial alcohol and cheese.
Abstract: A relatively simple, unstirred, non-aerated tank is the most common bioreactor in industrial use today. Many of the processes taking place in such reactors are anaerobic and are used to generate wine, potable and industrial alcohol and cheese. The processes are kinetically controlled and offer the potential of only limited productivity increases if more intensive mixing were to be utilised.