C. D. de Gooijer

Continuous production of baculovirus in a cascade of insect-cell reactors

SummaryInsect cells (Spodoptera frugiperda) were cultured in a continuous stirred-tank reactor. The effluent was led to a cascade of another two reactors, each containing half the volume of the cell-growth reactor, where the cells were infected with Autographa californica nuclear polyhedrosis virus. For about 10 days production of 107 polyhedra (virus particles embedded in a protein capsule) per cm3 was achieved. This short production time compared to previous experiments involving an analogous system with a single infection vessel of equal volume to the cell-growth vessel is ascribed to the accelerated occurence of the so-called passage effect (a decrease of infectious virus with time). From the results of a computer model it was concluded that this passage effect was accelerated by the change in residence time distribution as compared to the earlier experiments. time distribution as compared to the earlier experiments.

Bioreactors in series: An overview of design procedures and practical applications

Abstract “If one fermenter gives good results, two fermenters will give better results and three fermenters better still. This is sometimes true, but often false.” Herbert, D. 9

Dynamic modeling of immobilized Nitrosomonas europaea: Implementation of diffusion limitation over expanding microcolonies

Abstract A dynamic model (pseudohomogeneous growth model) describing growth and substrate consumption of immobilized Nitrosomonas europaea resulted in predictions for consumption rates that were higher than experimentally obtained data. This overestimation was caused by effects of diffusion limitation over microcolonies. Diffusion limitation over microcolonies has been integrated in the colony expansion model. In this model growth is implemented by expansion of colonies. Colonies breaking through the gel surface cause release of bacteria into the surrounding medium. Two options were considered for this release: either growth resulting in continuous leakage of single bacteria or eruption of entire colonies at once. In the latter case both macroscopic consumption rates and transient-state biomass concentration profiles are described satisfactorily by the model. It is also shown that the results of the previous experiments with Nitrobacter agilis can be described by the model as well.

A model for baculovirus production with continuous insect cell cultures.

SummaryA model, suitable for reactor-design purposes, is presented for the infection process of insect cells with baculovirus in a continuous bioreactor system. The infection process can be described by a first-order reaction mechanism if the viable, non-infected, insect cells are regarded as substrate, and the resulting polyhedra as product. Experimental results from continuous systems, consisting of a series of mixed reactors in which growth of Spodoptera frugiperda cells takes place in the first reactor and infection with Autographa californica nuclear polyhedrosis virus in the other reactors in the series, show typical values for the reaction rate constant of 0.7–1.0 10-5 s-1.

The effect of process conditions on the alpha-amylolytic hydrolysis of amylopectin potato starch: An experimental design approach.

The hydrolysis of amylopectin potato starch with Bacillus licheniformis alpha-amylase (Maxamyl) was studied under industrially relevant conditions (i.e. high dry-weight concentrations). The following ranges of process conditions were chosen and investigated by means of an experimental design: pH [5.6-7.6]; calcium addition [0-120 microg/g]; temperature [63-97 degrees C]; dry-weight concentration [3-37% [w/w]]; enzyme dosage [27.6-372.4 microL/kg] and stirring [0-200 rpm]. The rate of hydrolysis was followed as a function of the theoretical dextrose equivalent. The highest rate (at a dextrose equivalent of 10) was observed at high temperature (90 degrees C) and low pH (6). At a higher pH (7.2), the maximum temperature of hydrolysis shifted to a lower value. Also, high levels of calcium resulted in a decrease of the maximum temperature of hydrolysis. The pH, temperature, and the amount of enzyme added showed interactive effects on the observed rate of hydrolysis. No product or substrate inhibition was observed. Stirring did not effect the rate of hydrolysis. The oligosaccharide composition after hydrolysis (at a certain dextrose equivalent) did depend on the reaction temperature. The level of maltopentaose [15-24% [w/w]], a major product of starch hydrolysis by B. licheniformis alpha-amylase, was influenced mostly by temperature.

Design of aqueous two-phase systems supporting animal cell growth: a first step toward extractive bioconversions.

The design of aqueous two-phase systems (ATPSs) which support the long-term growth of animal cells is described in this paper. It was found that the increase in osmolality caused by the ATPS-forming polymers could be compensated by reducing the NaCl concentration of the culture medium. Cell growth was possible in culture media containing up to 0.025 g g-1 PEG or 0.15 g g-1 dextran. In ATPSs of PEG 35,000; dextran 40,000; and culture medium; the hybridoma cells partitioned to the PEG-lean phase. In two of these ATPSs, hybridoma cells were successfully cultured over a period of more than two months. The Mab product, however, partitioned along with the cells in the lower phase, but preliminary experiments using PEG ligands showed improved Mab partitioning. The design of aqueous two-phase systems (ATPSs) which support the long-term growth of animal cells is described in this paper. It was found that the increase in osmolality caused by the ATPS-forming polymers could be compensated by reducing the NaCl concentration of the culture medium. Cell growth was possible in culture media containing up to 0.025 g g-1 PEG or 0.15 g g-1 dextran. In ATPSs of PEG 35,000; dextran 40,000; and culture medium; the hybridoma cells partitioned to the PEG-lean phase. In two of these ATPSs, hybridoma cells were successfully cultured over a period of more than two months. The Mab product, however, partitioned along with the cells in the lower phase, but preliminary experiments using PEG ligands showed improved Mab partitioning.

Optimum design for a series of continuous stirred tank reactors containing immobilized biocatalyst beads obeying intrinsic Michaelis-Menten kinetics

The optimum design of a series of mixed reactors containing immobilized biocatalysts is described. A detailed description is given of the modelling of internal diffusion and reaction in the beads, and external mass transfer resistance. The model is validated by experiments with cascades of two and three reactors, containing immobilized invertase. For that, invertase was first bound to DE32-cellulose anion exchanger and then entrapped in Calcium Alginate.

Long-term semi-continuous production of recombinant baculovirus protein in a repeated (fed-)batch two-stage reactor system

Abstract The baculovirus expression system is commonly used in the research and development area and in the production of diagnostics and vaccines. Because the infection of insect-cell cultures with a (recombinant) baculovirus is a lytic process, the running time of an infected batch insect-cell reactor is limited. Another disadvantage of the system is the instability of the virus. In this study a two-stage reactor system was tested for its suitability for long-term semicontinuous operation. Three experimental setups were tested involving repeated infections in a reactor fed with cell suspension from a separate cell-growth reactor. Part of a previous infection was used as the virus inoculum. Best performance with respect to long-term operation was obtained with a repeated batch system. Twelve consecutive productive runs, consisting of infections during 5 days, could be performed. The titers of an infectious extracellular virus could be described well with an infection model previously developed in our laboratory.

Hybridoma and CHO Cell Partitioning in Aqueous Two-Phase Systems

The partitioning of mouse/mouse hybridoma cell line BIF6A7, mouse/rat hybridoma PFU‐83, and CHO DUKX B11‐derived cell line BIC‐2 in aqueous two‐phase systems (ATPSs) of poly(ethylene glycol) (PEG) and dextran was studied. The partitioning of BIF6A7 was investigated systematically by using a statistical experimental design. The aims were to identify the key factors governing cell partitioning and to select ATPSs with suitable cell partitioning for extractive bioconversions with animal cells. The influence of five factors, i.e., the poly(ethylene glycol) molecular weight (PEG MW), dextran molecular weight (Dx MW), tie‐line length (TLL), pH, and the ratio of potassium phosphate to potassium chloride, defined as the fraction KPi/(KPi + KCl), on BIF6A7 cell partitioning was characterized by using a full factorial experimental design. The cell partitioning ranged from complete partitioning into the interface to an almost complete partitioning to the lower phase. In all cases less than 1% of the cells partitioned to the top phase. The potassium phosphate fraction had the largest effect on cell partitioning. Low potassium phosphate fractions increased the proportion of cells in the lower phase. To a lesser extent the other factors also played a role in the cell partitioning. The best partitioning for the BIF6A7 cell line was obtained in ATPSs with PEG MW = 35 000, Dx MW = 40 000, TLL = 0.10 g/g, pH 7.4, and KPi/(KPi + KCl) = 0.1, where 93% of the cells were present in the lower phase. The previously reported partitioning of BIF6A7 cells in ATPS culture medium, corresponded well with the current findings. The partitioning of mouse/rat hybridoma cell line PFU‐83 and CHO cell line BIC‐2 was studied in an ATPS culture medium with PEG 35 000, dextran 40 000, TLL = 0.12 g/g, and hybridoma culture medium. Both cell lines partitioned almost completely into the lower phase. Moreover, the PFU‐83 cell line was able to grow in the ATPS hybridoma culture medium. This ATPS hybridoma culture medium therefore seems to be suitable for extractive bioconversions with a wide range of hybridoma cell lines, provided that their product can be partitioned into the upper PEG‐rich phase.

IgG and hybridoma partitioning in aqueous two-phase systems containing a dye-ligand

The effect of the important ATPS- and bufferparameters on IgG and hybridoma partitioning in ATPSscontaining a PEG-dye-ligand was studied. Objective wasto establish selection criteria for effective ligandsfor extractive fermentations with animal cells inATPSs.In the presence of 1% PEG-dye-ligand the binding ofIgG to the PEG-ligand was affected severely by theNa-chloride concentration. The tie-line length and pHaffected IgG partitioning to a lesser extent. Thedesired partitioning of IgG into the top phase, wasonly obtained when, in addition to the 10 mmol/kgK-phosphate buffer, no Na-chloride was present. In anATPS culture medium, with ± 35 mmol/kg Na-bicarbonateand 60 mmol/kg Na-chloride, increasing thePEG-dye-ligand concentration up to 100% did increasethe partition coefficient, but was not effective inconcentrating the IgG in the top phase of ATPS culturemedium at a pH of 7.8.Furthermore, addition of the PEG-dye-ligand to ATPSculture medium changed the hybridoma cell partitioningfrom the bottom phase to the interface.