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Biomedical subjects

J Tramper

Publications and source records attributed to J Tramper.

At least 73 records · Page 4Linked to original sources
PubMed1996

Automation of selective assays for on-line bioprocess monitoring by flow-injection analysis.

On-line analysis of one component in a complex media used for bioprocesses requires the application of selective tests such as enzymes assays. Because these assays are susceptible to interference by other medium components and have a limited detection range, automatic sample pretreatment is a prerequisite. The progress made with automatic sample pretreatment in flow-injection analysis makes this technique particularly suitable for on-line monitoring of bioprocesses. Moreover, newly developed software control systems may improve the necessary robustness of flow-infection analysis systems.

Animals↗
PubMed1996

Separation of hybridoma cells from their IgG product using aqueous two-phase systems.

The partitioning of IgG in aqueous two-phase systems (ATPSs) of PEG and Dextran was studied systematically using a statistical experimental design. Aim was to improve the separation of hybridoma cells and their IgG product, by identifying the key variables governing IgG partitioning, and by comparing the IgG partitioning data with the hybridoma cell partitioning data obtained in previous work. 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 potassium phosphate fraction (KPi/(KPi+KCl)), on IgG partitioning was characterized using a full-factorial experimental design. In all of the ATPS's the IgG partitioned predominantly into the lower phase. The partition coefficient varied between 0.78 (Variable settings: PEG Mw = 6000, Dx Mw = 500000, TLL = 0.10 g g-1, KPi/(KPi+KCl) = 1.0 and pH = 7.4) and 0.0002 (Variable settings: PEG Mw = 35000, Dx Mw = 40000, TLL = 0.20 g g-1 KPi/(KPi+KCl) = 1.0 and pH 6.6). The tie-line length, the dextran molecular weight and the PEG molecular weight had the most pronounced effect on IgG partitioning. Matching the partitioning data of the IgG product with previously obtained data of the hybridoma cell partitioning, showed that within the experimental design no ATPS could be found giving a good separation of the hybridoma cells and their IgG product. There are, however, ATPS's available in which the cells partition to, and grow in the lower dextran-rich phase. To achieve a good separation of the hybridoma cells and their IgG product in these ATPSs, the IgG product has to be specifically extracted into the PEG-rich top phase. For this purpose the use affinity ligands coupled to PEG may offer a solution. Therefore, a number of commercially available dye-resins was screened for their ability to bind the BIF6A7 IgG antibody. The mimetic green 1 A6XL dye-resin was found to bind BIF6A7 IgG. The dye-ligand coupled to PEG was used to manipulate the IgG partitioning in an ATPS. In the presence of the PEG-ligand, the IgG partitioned almost completely to the top phase. The IgG-partition coefficient increased three orders of magnitude, resulting in a 25-fold higher IgG concentration in the top phase than in the bottom phase.

Hybridomas↗
PubMed1995

On-line control of an immobilized hybridoma culture with multi-channel flow injection analysis.

An immobilized hybridoma cell line was cultivated at controlled glucose and glutamine concentrations. On-line analysis of the substrates was carried out with a multi-channel flow injection analysis system. The analysis system also determined on-line the lactate and ammonium concentration. The substrate concentrations were controlled using an adaptive-control strategy. This strategy consisted of the estimation of the real-time concentrations and volumetric substrate consumption rates by an Extended Kalman Filter, and a minimum variance controller, which used the estimated parameters to set the feed rates of the substrates. The closed-loop control was used to start-up two cultures with either glucose or glutamine as control-substrate for the medium feed rate. The controller kept the concentration of the control-substrate constant by enhancing the medium feed rate simultaneously to the increasing volumetric consumption rate of the substrate. When glutamine was used as control-substrate, the glucose concentration remained relatively constant, whereas the glutamine concentration decreased during the start-up at a constant glucose concentration. This indicates that glutamine is consumed faster than glucose and will be a better control-substrate to avoid limitation during the start-up of a culture with the applied hybridoma cell line. During the colonization of the microcarriers, the yield of ammonium on glutamine decreased from 0.80 to 0.55 (mol mol-1), indicating a change in the glutamine metabolism. The yield of lactate on glucose stayed constant for both experiments. During long-term culture of more than 800 h, the controller kept both the glucose and glutamine concentrations constant at perfusion rates between 0.50 h-1 and 0.15 h-1. The medium, glucose and glutamine feed rate were independently controlled. Both the specific glutamine and glucose consumption rates remained constant for all perfusion rates, which was probably as a result of the constant concentrations. The specific monoclonal antibody production rate decreased with the perfusion rate decreasing from 0.40 h-1 to 0.20 h-1. The immobilized-cell concentration decreased only at the lowest perfusion rate. Both effects could not be explained directly by the increasing ammonium and lactate concentrations nor by the decreasing amino-acid concentrations.

Animals↗
PubMed1995

Dextran as protectant against damage caused by sparging for hybridoma cells in a bubble column.

The effect of the addition of dextran as a protective polymer against sparging was examined with hybridoma suspension cells in a bubble column under standardized conditions. The protective effect of high concentrations of high molecular weight dextran showed a correlation with the bulk viscosity of the medium. A distinct protective effect occurs at viscosities greater than 20 x 10(-3) Pa s-1. In contrast, low molecular weight dextrans that cause a minor increase in viscosity, also provide no protection against sparging. There is no strict correlation between surface tension and the protective effect of dextran against sparging. Oxygen transfer is strongly reduced by high concentrations of high molecular weight dextran. Therefore, addition of dextran as protective polymer against sparging for large-scale production processes with animal cells in stirred reactors does not seem feasible.

Animals↗
PubMed1994

On-line monitoring of an animal cell culture with multi-channel flow injection analysis.

A multi-channel flow injection analysis system was used for on-line monitoring of a continuous animal cell culture with high cell density. With this system, the glucose, lactate and glutamine concentration were determined using immobilized dehydrogenases, ammonium using an aqueous o-phthaldialdehyde solution. Glutamine concentration was determined on the basis of the difference between a glutamine and a glutamate measurement. To prevent disturbance of the measurement and pollution of the system, the analytes in the sample were separated from high molecular compounds by on-line dialysis. On-line gas dialysis was used to avoid interference of other amino groups with the ammonium determination. In addition, dialysis was used as a dilution step. The measurement time for all four components was 42 min. This time included a final washing period after the analysis cycle. The system was calibrated once a day. Two continuous cultivations of a hybridoma cell line immobilized in open-porous glass carriers were monitored, using a fluidized bed reactor as cultivation system. The concentration of glutamine, glucose and ammonium determined with the on-line FIA system were in good agreement with the off-line data determined once a day. Only the lactate data showed some deviation. The immobilized enzyme reactors could be used for up to 3000-5000 injections. During the first cultivation, lasting 200 h, the start up period of the reactor was monitored. The on-line measurements described much better the time-course of the concentrations than the off-line data. It was possible to estimate the growth rate of the cells in the micro-carriers by the on-line data. In the course of the second cultivation, which lasted almost 1000 h, the influence of the dissolved oxygen concentration on the cell metabolism was monitored. It was noted that a sudden change of the glutamine concentration in the feed caused a fast change of the consumption and production rate of the measured metabolites.

Animals↗
PubMed1993

Location of two putative origins of DNA replication of Autographa californica nuclear polyhedrosis virus.

Previously, we described a defective Autographa californica nuclear polyhedrosis virus (AcMNPV) which must contain cis-acting elements required for DNA synthesis, such as the origin(s) of replication (ori). Defective genomes of AcMNPV generated after serial undiluted passage were analyzed further. Three small separated regions were retained in DNA of defective AcMNPV and accumulated in extracellular defective interfering viruses as well as in intracellular DNA after 40 passages. Two of these regions have now been identified as containing putative ori. They are located on the HindIII-B fragment between map units (m.u.) 50.1 and 53.2 and on the HindIII-Q fragment between m.u. 87.2 and 88.9 of the physical map of AcMNPV DNA, respectively. Transfection of Spodoptera frugiperda cells with plasmids containing these sequences followed by superinfection with intact helper AcMNPV resulted in amplification of these plasmids, as demonstrated by the Dpnl sensitivity assay. The replicating activity of HindIII-Q is putatively located within the 1000-bp region containing a highly repetitive DNA (hr5), which is also ascribed to enhance delayed-early gene expression. In order to demonstrate replicating activity of test plasmids, it appeared essential to transfect the cells well before superinfection with helper virus.

Animals↗
PubMed1993

Two-liquid-phase bioreactors.

The application of two liquid phases that are poorly miscible is a fascinating research topic for biocatalytical conversions because of the promising results. Motives for application include an increase of productivity and achievement of continuous processing, but new limitations arise, e.g., interfacial effects such as biocatalyst accumulation and loss of activity, medium component accumulation, and slow coalescence. Centrifuges, membranes, and immobilization are tools that can overcome part of the problems, but more fundamental knowledge about interfaces and coalescence is still necessary for successful application. For scaleup and further development of processes based on the obtained results, a choice must be made for the configuration of the experimental setup of a bioreactor. Aspects like aeration, shear stress, batch or continuous processing, and immobilization can play an important role. This review article describes these aspects and the proposals that have been made in recent years concerning two-liquid-phase bioreactors. It shows some adaptations to existing bioreactors, such as loop reactors and stirred-tank reactors.

Biodegradation, Environmental↗
PubMed1993

Identification of seven putative origins of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus DNA replication.

Seven putative origins of DNA replication (oris) were identified and located on the genome of Autographa californica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV), when an improved infection-dependent replication assay was used. A threefold higher yield of amplified plasmid was achieved when an m.o.i. of 1 was used (instead of 25), and another twofold increase was obtained when the interval between transfection and infection was extended from 5 to 24 h. Six of the putative oris were located in hr regions with homologous sequences. This suggests that all hrs in AcMNPV are bifunctional, i.e. have both ori and enhancer activity for transcription. In addition to the six hrs, the HindIII-K fragment of AcMNPV was also identified to carry a putative ori, although this fragment does not contain an hr region. However, the individual role of these seven oris during viral DNA replication, and whether they are all active simultaneously in vivo, is still unclear. The replication of an ori-containing plasmid starts at the same time (6 h post-infection) and proceeds at the same rate as viral DNA replication. A circular topology of ori-containing plasmids was a prerequisite for replication. Linear DNA, with an ori, did not replicate. Therefore, we suggest a theta structure or a rolling-circle as a model for baculovirus DNA replication.

Animals↗
PubMed1993

Quantitative Determination of the Spatial Distribution of Nitrosomonas europaea and Nitrobacter agilis Cells Immobilized in kappa-Carrageenan Gel Beads by a Specific Fluorescent-Antibody Labelling Technique.

A novel technique, combining labelling and stereological methods, for the determination of spatial distribution of two microorganisms in a biofilm is presented. Cells of Nitrosomonas europaea (ATCC 19718) and Nitrobacter agilis (ATCC 14123) were homogeneously distributed in a kappa-carrageenan gel during immobilization and allowed to grow out to colonies. The gel beads were sliced in thin cross sections after fixation and embedding. A two-step labelling method resulted in green fluorescent colonies of either N. europaea or N. agilis in the respective cross sections. The positions and surface areas of the colonies of each species were determined, and from that a biomass volume distribution for N. europaea and N. agilis in kappa-carrageenan gel beads was estimated. This technique will be useful for the validation of biofilm models, which predict such biomass distributions.

Journal Article↗
PubMed1993

Simultaneous nitrification and denitrification using immobilized microorganisms.

Nitrogen removal from wastewaters is a multiple step process in which nitrification is often a problem due to the slow growth rates of the nitrifying bacteria. By immobilization of these bacteria, nitrification can be efficiently accomplished in compact reactors. In this paper, the possibilities of integrated nitrification and denitrification within a single reactor system are evaluated. Two main systems are studied: a) Nitrosamines europaea and Pseudomonas denitrificans co-immobilized in a a gel bead and operated in an air-lift reactor; b) the same bacteria separately immobilized and operated in different compartments of a multiple gas-lift reactor.

Ammonia↗
PubMed1992

Continuous beta-galactosidase production with a recombinant baculovirus insect-cell system in bioreactors.

Insect cells were exploited to produce bacterial beta-galactosidase by infecting them with a recombinant nuclear polyhedrosis virus (baculovirus) of Autographa californica. The insect cells were cultured in a continuous stirred tank reactor (CSTR) and led to a second CSTR where they were infected with a recombinant virus in which the lacZ gene from Escherichia coli was inserted. In the effluent of the production reactor, maximum activities of 15 units beta-galactosidase per 10(6) cells were measured. For about 25 d beta-galactosidase production remained constant, but then rapidly declined. This drop was due to a decrease in production of active beta-galactosidase rather than to inactivation of this enzyme. It was concluded that the reduced production was due to reduced polyhedrin promoter-driven synthesis.

Animals↗
PubMed1991

Detection and analysis of Autographa californica nuclear polyhedrosis virus mutants with defective interfering properties.

Defective interfering particles (DIPs) were generated upon continuous production of Autographa californica nuclear polyhedrosis virus (AcNPV) in bioreactors. This configuration mimicked the serial undiluted passaging of virus, which is known to result in plaque-morphology mutants. Restriction enzyme analysis of DIP-containing preparations of extracellular virus showed the presence of many DNA fragments in less than equimolar amounts. These fragments were colinear on the physical map of AcNPV and extended from map position 1.7 to 45. These DIPs thus lacked 43% of the genetic information of the standard virus, including the polyhedrin and DNA polymerase genes. The existence of DIPs was confirmed by electron microscopy, where virions were observed with reduced length. Among the less than equimolar fragments in DIP-containing preparations, fragments were observed linking sequences from map positions 1.7 and 45 via a TGTT linker of unknown origin. The DIPs could not be plaque-purified and needed standard (helper) virus to replicate; DIP-containing preparations interfered with standard virus replication in an interference assay, which explained the reduction in productivity of an AcNPV expression vector-insect cell system in continuous bioreactor operations. The origin of these DIPs and their possible generation mechanism are discussed.

Animals↗
PubMed1990

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

Insect 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 10(7) 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 occurrence 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.

Animals↗
PubMed1979

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.

Animals↗
PubMedDate not supplied

Metabolite-balancing techniques vs. 13C tracer experiments to determine metabolic fluxes in hybridoma cells.

The estimation of intracellular fluxes of mammalian cells using only mass balances of the relevant metabolites is not possible because the set of linear equations defined by these mass balances is underdetermined. In order to quantify fluxes in cyclic pathways the mass balance equations can be complemented with several constraints: (1) the mass balances of co-metabolites, such as ATP or NAD(P)H, (2) linear objective functions, (3) flux data obtained by isotopic-tracer experiments. Here, these three methods are compared for the analysis of fluxes in the primary metabolism of continuously cultured hybridoma cells. The significance of different theoretical constraints and different objective functions is discussed after comparing their resulting flux distributions to the fluxes determined using 13CO2 and 13C-lactate measurements of 1 - 13C-glucose-fed hybridoma cells. Metabolic fluxes estimated using the objective functions "maximize ATP" and "maximize NADH" are relatively similar to the experimentally determined fluxes. This is consistent with the observation that cancer cells, such as hybridomas, are metabolically hyperactive, and produce ATP and NADH regardless of the need for these cofactors.

Adenosine Triphosphate↗
PubMedDate not supplied

Field-emission scanning electron microscopy analysis of morphology and enzyme distribution within an industrial biocatalytic particle.

Field-emission scanning electron microscopy (FESEM) was used in a technical feasibility study to obtain insight into the internal morphology and the intraparticle enzyme distribution of Assemblase, an industrial biocatalytic particle containing immobilized penicillin-G acylase. The results were compared with previous studies based on light and transmission electron microscopic techniques. The integrated FESEM approach yielded the same quantitative results as the microscopic techniques used previously. Given this technical equivalence, the integrated approach offers several advantages. First, the single preparation method and detection system avoids interpretation discrepancies between corresponding areas that were examined for different properties with different detection techniques in different samples. Second, the specimen size suitable for whole particle study is virtually unlimited, which simplifies sectioning and puts less stringent demands on the embedding technique. Furthermore, the sensitivity toward enzyme presence and distribution increases because the epitopes inside thick sections become available for labeling. Quick and unambiguous analysis of the relation between particle morphology and enzyme distribution is important because this information may be used in the future for the design of enzyme distributions in which the particle morphology can be used as a control parameter.

Drug Industry↗