Showing papers by "Paul F. Greenfield published in 1994"
TL;DR: Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large‐scale systems for the production of recombinant products using the baculovirus expression vector system.
Abstract: A mathematical model has been developed that predicts the cell population dynamics and production of recombinant protein and infective extracellular virus progeny by insect cells after infection with baculovirus in batch suspension culture. Infection in the model is based on the rate of virus attachment to suspended insect cells under culture conditions. The model links the events following infection with the sequence of gene expression in the baculovirus replicative cycle. Substrate depletion is used to account for the decrease in product yield observed when infecting at high cell densities. Model parameters were determined in shaker flasks for two media: serum-supplemented IPL-41 medium and serum free Sf900II medium. There was good agreement between model predictions and the results from an independent series of experiments performed to validate the mode. The model predicted: (1) the optimal time of infection at high multiplicity of infection: (2) the timing and magnitude of recombinant protein production in a 2-L bioreactor; and (3) the timing and magnitude of recombinant protein production at multiplicities of infection from 0.01 to 100 plaque-forming units per cell. Through its ability to predict optimal infection strategies in batch suspension culture, the model has use in the design and optimization of large-scale systems for the production of recombinant products using the baculovirus expression vector system.
85 citations
TL;DR: In this paper, the overall performance of a two-phase anaerobic wastewater treatment system was determined as a function of the extent of recycle of the effluent from the methanogenic reactor to the acidification reactor.
64 citations
TL;DR: Oxygen uptake rates (THE AUTHORS') of Sf9 insect cells propagated in a serum-free medium and of cells infected with a recombinant AcNPV were investigated before and after infection in a laboratory-scale bioreactor and it is suggested that THEIR measurement can be used to reflect the efficiency of a batch infection.
Abstract: Oxygen uptake rates (OUR) of Sf9 insect cells propagated in a serum-free medium (SF900II, Gibco) and of cells infected with a recombinant AcNPV were investigated before and after infection in a laboratory-scale bioreactor. The volumetric OURs of uninfected and exponentially growing cells were found to be proportional to the cell density. For infected cultures, the specific OUR of cells increased immediately after addition of virus and a maximum of 1.3 times the value of uninfected cells was noted for all the cultures between 8 to 30 hours post infection, which coincides with the period at which most viral replication and the majority of DNA synthesis takes place. It was observed that the rate of rise in the specific OUR decreased as the cell density at the time of infection increased, which meant that the later the infection, the later the maximum sOUR was observed. We therefore suggest that OUR measurement can be used to reflect the efficiency of a batch infection. Carbohydrate and amino acid consumption rates from an infected run were analysed in an effort to identify substrate(s) that may be used at increased rates to fuel the rise in oxygen demand observed early in the infection cycle. No observable rise in the consumption rates of glucose or glutamine, which are the major energy sources for animal cells, were seen after infection but an increase in the consumption rates of some amino acids suggests that infected Sf9 cells may utilise amino acids at an enhanced rate for energy post infection.
60 citations
TL;DR: The nature and temporal development of ammonia inhbition were investigated in batch, fed-batch, and continuous cultures, and significant inhibition was observed when cells were inoculated in serum-containing or chemically defined medium containing more than 2 mM of ammonia.
Abstract: The nature and temporal development of ammonia inhibition were investigated in batch, fed-batch, and continuous cultures. Significant inhibition was observed when cells were inoculated in serum-containing or chemically defined medium containing more than 2 mM of ammonia. In contrast, no inhibition was observed at greater than 10 mM when the ammonia concentration was gradually increased over the span of a batch culture by feeding ammonium chloride. Strong growth inhibition was observed after each of five step changes (2.8 --> 3.7 --> 4.0 --> 4.9 --> 7.7 --> 13.5 mM) in continuous culture. Following a period of adaptation at each higher value, the viable cell density stabilized at a new lower value. The lowering in viable cell density was caused by an increase in specific death rate and a decreased cell yield on glucose, glutamine, and oxygen. Increased ammonia concentration had little or no effect on the steady-state specific growth kinetics or specific antibody productivity. (C) 1994 John Wiley and Sons, Inc.
39 citations
TL;DR: In this article, two laboratory scale Sequencing Batch Reactors (SBR) were fed with wastewater from two different stages of anaerobic pretreatment using waste stabilization ponds.
28 citations
TL;DR: In this article, the effect of lowering the pH of the acidification reactor on the overall performance of a two-stage anaerobic wastewater treatment system was determined both experimentally and through simulation of a dynamic structured model.
15 citations
TL;DR: The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor and significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were observed.
Abstract: A composite gel system has been developed combining the chemical and physical properties of calcium alginate and agarose gels. The results of growing composite gel immobilized hybridoma SPO1 cells in a protein-free medium within a fluidized-bed perfusion bioreactor are presented in this paper. During the continuous operation of this system, the total cell density reached 3.9×107 cells per ml of beads (viability 79.6%). The specific productivity of monoclonal antibody of the immobilized hybridoma cells reached more than 1.5 μg per 106 viable cells per hour, compared with 0.5 for non-immobilized viable cells grown in a one liter agitated bioreactor with the same medium. Significant increases in cell metabolic activities, including substrate utilization and byproduct formation, were also observed. Leaching of materials from the beads was evident and the major fraction of released materials was alginate.
13 citations
TL;DR: The immobilization process using small beads caused a lower cell viability initially but allowed a higher rate of cell growth subsequently, compared to those in large beads, and a significant drop in the formation of lactate after the cell growth entered a steady state suggested a higher activity of the Tricarboxylic Acid Cycle in the cells when the cell density became high.
Abstract: Hybridoma SPO1 cells were immobilized in calcium alginate beads and were further grown in a fluidized-bed perfusion system with a protein-free medium. The presence of serum in the steps of entrapment was shown to be helpful for the preservation of cell viability. Each step during immobilization was investigated with respect to the extent of cell damage caused. The immobilization process using small beads caused a lower cell viability initially but allowed a higher rate of cell growth subsequently, compared to those in large beads. In a perfusion system for the continuous production of monoclonal antibodies (MAb), the viable cell density reached 2×107 cells per ml of beads with a viability of 40%. Compared with the cells in suspension culture, the immobilized SPO1 cells showed higher viable cell based specific rates of substrate uptake (glucose and glutamine) and of MAb production. A significant drop in the formation of lactate after the cell growth entered a steady state suggested a higher activity of the Tricarboxylic Acid Cycle in the cells when the cell density became high.
13 citations
TL;DR: Optimisation of the glucose feed rate and feed concentration for the production of a recombinant protein (β-galactosidase) with respect to two different objective functions is addressed in this work.
6 citations
TL;DR: In this article, an assessment was made of biological phosphorus and nitrogen removal and related sludge bulking control in both intermittent cyclic and continuous activated sludge systems, and the results from a laboratory-scale investigation using fermented domestic sewage indicated the advantages of achieving high levels of P and N removal and good sludge-settling properties using the intermittently cyclic process, as compared to the continuous process.
Abstract: An assessment was made of biological phosphorus and nitrogen removal and related sludge bulking control in both intermittent cyclic and continuous activated sludge systems. Experimental results from a laboratory-scale investigation using fermented domestic sewage indicated the advantages of achieving high levels of P and N removal and good sludge-settling properties using the intermittent cyclic process, as compared to the continuous process. The modified intermittently fed and decanted system incorporating non-mixing sequences produced an effluent quality of NO3-N 60 mg L-1, TP-9 mg L-1 and RBCOD < 30 mg L-1). In contrast, the modified 4-stage Bardenpho process could not achieve effluent NO3-N and PO4-P concentrations of < 10 and < 1 mg L-1, respectively. The sludge volume index (SVI) values were always higher than 250 mL g-1 and occasionally reached as high as 422 mL g-1. The causative filaments were low F:M growers - Types 0041/0675.
6 citations
TL;DR: It will be shown that the model quantitatively describes the trade off between cell and product yield observed at high cell density by depletion of a limiting substrate.
Abstract: An unstructured mathematical model has been developed in our laboratory to describe the growth and infection of sf9 insect cells by a recombinant Baculovirus (pAc-360 β-Gal). The model quantitatively predicts the effects of altering the three key optimisation parameters, time of infection (TOI), multiplicity of infection (MOI) and time of harvest (TOH), in two different media, IPL-41+5% FCS+0.1% Pluronic F68 and Sf900II (Gibco). Here, the model is described for the limiting case of infection of sf9 cells in IPL-41+5% FCS+0.1% Pluronic F68 medium with a high multiplicity of infection, ie. the case of synchronous infection. Using parameters obtained from a series of independent experiments, it will be shown that the model quantitatively describes the trade off between cell and product yield observed at high cell density by depletion of a limiting substrate.
01 Jan 1994
TL;DR: The proportionality between viable cell density and the volumetric oxygen uptake rate (vOUR) indicates that vOUR can be used as an online measurement of viable celldensity enabling automatic infection, and high yielding infections were characterised by a continued significant increase in volumetrics uptake rate following infection.
Abstract: In this article, the problem of optimising insect cell cultures infected with a recombinant Baculovirus is discussed. A model of the infection of insect cells in batch cultures suggests that the maximum achievable yield is very sensitive to the time of infection (TOI). The optimal TOI is a function of the multiplicity of infection (MOI). MOI itself, however, has little effect on the maximum achievable yield. The optimal time of infection was found to be during the early to mid-exponential phase of the culture. The specific oxygen uptake rate was found to be at its highest and to be relatively constant during this period. The proportionality between viable cell density and the volumetric oxygen uptake rate (vOUR) indicates that vOUR can be used as an online measurement of viable cell density enabling automatic infection. Furthermore, high yielding infections were characterised by a continued significant increase in volumetric oxygen uptake rate following infection. This indicates that vOUR also has potential as a diagnostic tool.
01 Jan 1994
TL;DR: Low cell density, suboptimal nutrient environment and increased down time, and labour of repeated batch fermentations impose major limitations to high product yields in recombinant baculovirus infected insect cell cultures.
Abstract: Low cell density, suboptimal nutrient environment and increased down time, and labour of repeated batch fermentations impose major limitations to high product yields in recombinant baculovirus infected insect cell cultures.
01 Jan 1994
TL;DR: In this article, an improved version of a mechanistic mathematical model of anaerobic degradation processes has been developed, which was incorporated into three types of high-rate reactor configuration; namely, single-stage, two-stage and two stage with recycle.
Abstract: An improved version of a mechanistic mathematical model of anaerobic degradation processes has been developed. It was incorporated into three types of high-rate reactor configuration; namely, single-stage, two-stage, and two-stage with recycle. Implemented in a recently developed simulation package NIMBUS (1) the model was able to simulate the dynamic behaviour of anaerobic wastewater treatment systems. In the present study, the model has been used to predict the system response to a short-term concentration and hydraulic step changes at various volumetric organic loading rates. The comparison between the predicted and the measured data using a synthetic diluted molasses wastewater showed that reactor variables such as pH, alkali consumption rate, gas production and its composition, and effluent organic acids concentration were modelled reasonably well.