Manfred Biselli

Dielectric spectroscopy in the cultivation of suspended and immobilized hybridoma cells.

Dielectric spectroscopy, also referred to as capacitance measurement, was evaluated as a tool for on-line and real-time monitoring of hybridoma cell growth in suspension batch culture and in immobilized cell culture in porous glass carriers. The capacitance signal proved to be a lumped parameter influenced by cell concentration, cell size and culture conditions. During a batch culture the cell specific capacitance signal changes by about 45% having a maximum value at the maximum growth rate. An excellent correlation between the specific capacitance and the specific amount of nucleotidetriphosphates in the cells could be shown. Dielectric spectroscopy proved to be a useful tool for on-line monitoring of cell attachment and growth in open porous microcarriers in fluidized bed fermenters. Also, in this system only an approximate correlation with viable cell concentration appeared, whereas an exact correlation with the glutamine consumption rate, a measure of the metabolic activity of the cells, could be shown. This allowed a closed loop control of the medium feed rate, which was directly linked to the capacitance signal during the entire course of a continuous fermentation.

Development of a fixed bed bioreactor for the expansion of human hematopoietic progenitor cells

The ex vivo expansion of hematopoietic progenitor cells is of great interest for a variety of clinical applications, e.g. bone marrow transplantation or gene therapy. Therefore it is of general interest to develop a culture system, able to mimic the in vivo hematopoesis, which is a prerequisite for long-term hematopoietic culture. Our approach was to modify a continuously perfused bioreactor for cultivation and expansion of human hematopoietic stem cells. Therefore we immobilized stromal cells (human primary stromal cells or the murine cell line M2-10B4) in porous glass carriers in a fixed bed reactor and cocultivated human hematopoietic progenitor cells for several weeks. After inoculation of mononuclear cells derived from umbilical cord blood or peripheral blood stem cells both adherent and non adherent cells were harvested and analyzed by flow cytometry and short-term colony assays. During cultivation there was a permanent production of progenitor cells and mature blood cells derived from the immobilized cells in the carriers. We could demonstrate the immobilization of hematopoietic progenitor cells of the myeloid system detectable in short-term colony assays. Additionally we could observe the expansion of very early progenitor cells (CFU-GEMM) up to 4.2-fold and later progenitor cells (CFU-GM and BFU-E) up to 7-fold and 1.8-fold, respectively.

Cultivation of Hematopoietic Stem and Progenitor Cells: Biochemical Engineering Aspects

The ex vivo expansion of hematopoietic cells is one of the most challenging fields in cell culture. This is a rapidly growing area of tissue engineering with many potential applications in bone marrow transplantation, transfusion medicine or gene therapy. Over the last few years much progress has been made in understanding hematopoietic differentiation, discovery of cytokines, isolation and identification of cellular subtypes and in the development of a variety of bioreactor concepts. All this has led to a number of (preliminary) clinical trials that gave a hint of the benefits that can be obtained from the use of expanded hematopoietic cells in therapy. Moreover, as we understand the complexity and the regulation of hematopoiesis, it becomes obvious that highly sophisticated cultivation techniques and bioreactor concepts are needed: a new challenge for bioprocess engineering in cell culture.

The influence of dissolved oxygen tension on the metabolic activity of an immobilized hybridoma population

In the study presented here a laboratory scale (150 ml) fluidized bed bioreactor was used as a tool for making kinetic measurements on the production of monoclonal antibodies (MAb) with a hybridoma cell line. We determined the influence of dissolved oxygen tension (DOT) on the metabolic activity of a hybridoma population immobilized in macroporous collagen microspheres. The data obtained showed a reduction of the metabolic activity of the immobilized population at reduced DOT, the total number of immobilized cells, however, remained constant. At decreasing DOT an increasing lactate yield from glucose at reduced glutamine consumption was noticed, indicating a shift in the pattern of substrate usage. A mathematical description of maintenance metabolism was formulated and the parameters of growth and maintenance requirements were calculated. A growth associated MAb production was determined under the conditions applied leading to space time yields of 225 mg MAb per liter of total reactor volume and day.

Chip-based amperometric enzyme sensor system for monitoring of bioprocesses by flow-injection analysis.

A microfluidic chip integrating amperometric enzyme sensors for the detection of glucose, glutamate and glutamine in cell-culture fermentation processes has been developed. The enzymes glucose oxidase, glutamate oxidase and glutaminase were immobilized by means of cross-linking with glutaraldehyde on platinum thin-film electrodes integrated within a microfluidic channel. The biosensor chip was coupled to a flow-injection analysis system for electrochemical characterization of the sensors. The sensors have been characterized in terms of sensitivity, linear working range and detection limit. The sensitivity evaluated from the respective peak areas was 1.47, 3.68 and 0.28 μAs/mM for the glucose, glutamate and glutamine sensor, respectively. The calibration curves were linear up to a concentration of 20 mM glucose and glutamine and up to 10 mM for glutamate. The lower detection limit amounted to be 0.05 mM for the glucose and glutamate sensor, respectively, and 0.1 mM for the glutamine sensor. Experiments in cell-culture medium have demonstrated a good correlation between the glutamate, glutamine and glucose concentrations measured with the chip-based biosensors in a differential-mode and the commercially available instrumentation. The obtained results demonstrate the feasibility of the realized microfluidic biosensor chip for monitoring of bioprocesses.

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.

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.

An approach to integrated antibody production: Coupling of fluidized bed cultivation and fluidized bed adsorption

Continuous culture may be an efficient way of producing proteins which are susceptible to secondary processing in the course of a fermentation process. Short residence times in these systems support the production of correctly assembled proteins by avoiding substrate limitations and product inhibitions and also minimize the contact of sensitive bioproducts with degrading enzymes. Thus products of increased stability and integrity are obtained from continuous processes. The downstream process following continuous culture has to be adapted to the specific conditions of continuous fermentations, e.g. large liquid volumes and diluted process solutions. In this paper an approach is shown how a fluidized bed adsorption as first recovery operation may be coupled directly to a continuous production. Immobilized hybridoma cells are cultivated in porous glass microcarriers in a continuous fluidized bed process, the cell containing harvest is purified by fluidized bed adsorption using an agarose based cation exchange matrix. By this coupled mode of operation the large biomass containing harvest volume resulting from the continuous cultivation may be applied directly to a fluidized chromatographic matrix without prior clarification, leading to a particle free and initially purified product solution of reduced volume. In an experimental setup a bench-scale fluidized bed bioreactor of 25 ml carrier volume was coupled to a fluidized bed adsorption column operated with 300 ml of adsorbent. This configuration yielded up to 20 mg of monoclonal antibody per day in a cell free solution at fourfold concentration and fivefold purification. The process was run for more than three weeks with consistent product output.

Preparation of cellodextrins and isolation of oligomeric side components and their characterization

Cellodextrin (beta-1,4-glucose oligomer) mixtures are prepared by precipitation of oligomers with 1-propanol and ethanol after partial hydrolysis of cellulose with hydrochloric acid or by acetolysis of cellulose. Cellooligomers (DP3-DP8) can be isolated by high-resolution size-exclusion chromatography on Bio-Gel P 4 using water as eluent. Recycle operation of the columns allows the separation of oligomers up to a degree of polymerization of 12. However, ion-exchange chromatography of their borate complexes demonstrates the heterogeneity of cellodextrins, homogeneous according to size-exclusion chromatography. At least four secondary oligomeric components are observed in the different samples. By preparative affinity chromatography on phenyl-boronate-agarose two of these components could be purified and subsequently characterized. In one series of oligosaccharides the glucose unit at the reducing end of the beta-1,4-glucose oligomers is derivatized to fructose. This enolization reaction occurs during size-exclusion chromatography. The precipitation step with alkanols during preparation of oligomer mixtures generates oligomeric glycosides. Additionally, the formation of amines from respective beta-1,4-glucose oligomers is observed with the ammonium carbonate eluent used in affinity chromatography. Analysis methods combined to assess for the homogeneity of cellodextrins include enzyme- and acid-catalyzed (partial) hydrolysis of the different oligomers and subsequent analysis of degradation products by sugar borate chromatography; 13C and 1H NMR spectroscopy; and fast atom bombardment mass spectroscopy.

Tools and applications of biochemical engineering science

F. Tholey, E. Heinzle: Methods for Biocatalyst Screening.- E. Stark, B. Hitzmann, K. Schugerl, T. Scheper: In-Situ-Fluorescence-Probes: A Useful Tool for Non-Invasive Bioprocess Monitoring.- C. Wittmann: Metabolic Flux Analysis Using Mass Spectrometry.- S. Nath: The Molecular Mechanism of ATP Synthesis by F1F0-ATP Synthase: A Scrutiny of the Major Possibilities.- I. Jasmund, A. Bader: Bioreactor Developments for Tissue Engineering Applications by teh Example of the Bioartificial Liver.- t. Noll, N. Jelinek, S. Schmidt, M. Biselli, C. Wandrey: Cultivation of Hematopoietic Stem and Progenitor Cells: Biochemical Engineering Aspects.- L.R. Castilho, R.A. Medronho: Cell Retention Devices for Suspended-Cell Perfusion Cultures.- N. Chang, S.J. Han, S.C. Inm, M.-R. Han, J. Lee: Fed-Batch Cultures of Escherichia coli Cells with Oxygen-Dependent nar Promoter Systems.- L. Maranga, P.E. Cruz, J.G. Aunins, M.J.T. Carrondo: Production of Core and Virus-Like Particles With Baculovirus Infected Insect Cells.- I. Wagner-Dobler, W. Beil, S. Lang, M. Meiners, H. Laatsch: Integrated Approach to Explore the Potential of Marine Microorganisms for the Production of Bioactive Metabolites.- A.-P. Zeng, H. Biebl: Bulk-Chemicals from Biotechnology: The Case of 1,3-Propanediol Production and the New Trends.