Showing papers on "Bioprocess published in 2006"

Operational strategies, monitoring and control of heterologous protein production in the methylotrophic yeast Pichia pastoris under different promoters: A review

Abstract: The methylotrophic yeast Pichia pastoris has been widely reported as a suitable expression system for heterologous protein production. The use of different phenotypes under PAOX promoter, other alternative promoters, culture medium, and operational strategies with the objective to maximize either yield or productivity of the heterologous protein, but also to obtain a repetitive product batch to batch to get a robust process for the final industrial application have been reported. Medium composition, kinetics growth, fermentation operational strategies from fed-batch to continuous cultures using different phenotypes with the most common PAOX promoter and other novel promoters (GAP, FLD, ICL), the use of mixed substrates, on-line monitoring of the key fermentation parameters (methanol) and control algorithms applied to the bioprocess are reviewed and discussed in detail.

Real-time bioprocess monitoring: Part I: In situ sensors

Abstract: In the past decades, biotechnology has exploded to a cutting edge industry and its economical significance is exponentially growing. Bioprocess efficiency, productivity and optimization are essential. Efficient process control allows quality of the product to be maintained, but also reduces costs, increases profit, and reduces pollution of the environment. Real-time process monitoring is fundamental for good process control. Rapid development of bioprocess applications led to intensive search for new sensors able to provide real-time information about the process. Real-time sensors may operate in an in situ (in-line) and ex situ on-line set-up. This work reviews the approaches that have been used for bioprocess monitoring and is divided in two parts. First part deals with in situ methods with sensors placed inside the reactor and in direct contact with bulk medium. Second part discusses sample handling techniques (e.g. FIA and SIA) that allow sample withdrawal from the reactor and automatic handling, transport to the sensor, and ex situ, on-line monitoring.

Pulsed Electric Fields (PEF) for Permeabilization of Cell Membranes in Food- and Bioprocessing – Applications, Process and Equipment Design and Cost Analysis.

Abstract: The impact of pulsed electric fields (PEF) on phospholipid vesicles, plant and animal as well as microbial and protozoa membranes was investigated. A series of pulse modulators and treatment chambers was realized in order to examine the diversity of components, materials and processing parameters. Electric field strength, energy input and treatment temperature were identified as key processing parameters. A critical field strength of 0.3 to 0.5 kV/cm for plant and animal and 10 to 15 kV/cm for microbial cells was observed. Degree of permeabilization was investigated by impedance analysis for plant and animal tissue and flow cytometry for microbes and liposomes to optimize processing parameters. The impact of membrane permeabilization on mass transfer processes was investigated for plant and animal tissue in laband technical scale. It was shown that extractability of fruit and vegetable juices or intracellular compounds can be enhanced after a PEF-treatment. An increase of up to 7 % of yield was found in comparison to untreated samples, juice quality was equivalent. Technical-scale treatments revealed the impact of a PEF-treatment on structural properties of fruit mash, an adaptation of liquid-solid separation techniques was shown to be required. A PEF-treatment of meat resulted in enhanced mass transfer during drying as well as brining of products, an improvement of water binding during cooking was found due to improved microdiffusion of brine and water binding agents. Microbial inactivation was investigated in different liquid media. For fruit juice and milk the applicability to achieve a gentle preservation was shown. The impact of processing parameters was evaluated in order to reduce electric energy requirements. A combined application of PEF and mild heat showed highly synergetic effects and improved energy efficiency. Enzyme inactivation was determined for lactoperoxidase in milk in comparison to thermal inactivation. It was observed that only a minor part of the inactivation was related to electric field effects, whereas at higher treatment intensities mainly thermal effects occurred. In addition the PEF applicability to achieve disintegration of sludge during waste water processing and for preservation of algae extracts was shown. Energy requirements to induce pore formation in different biological membrane systems were compared dependent on transmembrane potential induced. An analysis of cost efficiency showed that disintegration of plant and animal material by PEF is superior in comparison to a conventional treatment in terms of energy and time requirements as well as costs of operation. For microbial inactivation by PEF even an optimized treatment resulted in higher production costs, but consumer and quality benefits might justify these extra efforts for premium or thermally sensitive products. Meat, fruit and vegetable treatment were identified as the most promising applications to achieve a broad industrial exploitation of the technique, approximately 50 years after first empirical reports by Heinz Doevenspeck. Acknowledgements III

Engineered bacteriophage-defence systems in bioprocessing

Abstract: Bacteriophages (phages) have the potential to interfere with any industry that produces bacteria as an end product or uses them as biocatalysts in the production of fermented products or bioactive molecules. Using microorganisms that drive food bioprocesses as an example, this review will describe a set of genetic tools that are useful in the engineering of customized phage-defence systems. Special focus will be given to the power of comparative genomics as a means of streamlining target selection, providing more widespread phage protection, and increasing the longevity of these industrially important bacteria in the bioprocessing environment.

An aqueous–organic two-phase bioprocess for efficient production of the natural aroma chemicals 2-phenylethanol and 2-phenylethylacetate with yeast

Abstract: The natural aroma chemicals 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc) are of high industrial relevance and can be produced from L-phenylalanine in a yeast-based process with growth-associated product formation. Due to product inhibition, in situ product removal is mandatory to obtain economically interesting concentrations. A fed-batch approach using polypropylene glycol 1200 as in situ extractant and the precursor in a saturated concentration led to the highest 2-PE productivity reported for a bioprocess so far. With Kluyveromyces marxianus CBS 600, 26.5 g/l 2-PE and 6.1 g/l 2-PEAc in the organic phase were obtained, corresponding to space-time yields of 0.33 and 0.08 g/l h, respectively.

Improvement of bioprocess monitoring: development of novel concepts

Abstract: The advancement of bioprocess monitoring will play a crucial role to meet the future requirements of bioprocess technology. Major issues are the acceleration of process development to reduce the time to the market and to ensure optimal exploitation of the cell factory and further to cope with the requirements of the Process Analytical Technology initiative. Due to the enormous complexity of cellular systems and lack of appropriate sensor systems microbial production processes are still poorly understood. This holds generally true for the most microbial production processes, in particular for the recombinant protein production due to strong interaction between recombinant gene expression and host cell metabolism. Therefore, it is necessary to scrutinise the role of the different cellular compartments in the biosynthesis process in order to develop comprehensive process monitoring concepts by involving the most significant process variables and their interconnections. Although research for the development of novel sensor systems is progressing their applicability in bioprocessing is very limited with respect to on-line and in-situ measurement due to specific requirements of aseptic conditions, high number of analytes, drift, and often rather low physiological relevance. A comprehensive survey of the state of the art of bioprocess monitoring reveals that only a limited number of metabolic variables show a close correlation to the currently explored chemical/physical principles. In order to circumvent this unsatisfying situation mathematical methods are applied to uncover "hidden" information contained in the on-line data and thereby creating correlations to the multitude of highly specific biochemical off-line data. Modelling enables the continuous prediction of otherwise discrete off-line data whereby critical process states can be more easily detected. The challenging issue of this concept is to establish significant on-line and off-line data sets. In this context, online sensor systems are reviewed with respect to commercial availability in combination with the suitability of offline analytical measurement methods. In a case study, the aptitude of the concept to exploit easily available online data for prediction of complex process variables in a recombinant E. coli fed-batch cultivation aiming at the improvement of monitoring capabilities is demonstrated. In addition, the perspectives for model-based process supervision and process control are outlined.

Near-infrared spectroscopic monitoring of biomass, glucose, ethanol and protein content in a high cell density baker's yeast fed-batch bioprocess.

Abstract: The use of at-line NIRS to monitor a high cell density fed-batch baker's yeast bioprocess was investigated. Quantification of the key analytes (biomass, ethanol and glucose) and the product quality indicator (percentage protein content) was studied. Biomass was quantitatively modelled using whole matrix samples (as was percentage protein content). The dominance of the whole matrix spectrum by biomass, and its associated light scattering effects, were overcome by use of filtrate samples and adapted (semi-synthetic) filtrate samples, which allowed successful ethanol and glucose modelling, respectively. Calibrations were rigorously challenged via external validation with large sample sets relative to the calibration sample size, ensuring model robustness and potential practical utility. The standard errors of calibration for biomass, glucose, ethanol and total intracellular protein were (g/l) 1.79, 0.19, 0.79 and 0.91, respectively, comparable to those of the primary assays. The calibration strategies necessary to generate quantitative models for this range of analytes in such a complex high cell density bioprocess fluid are discussed.

Scaled-up production of mammalian neural precursor cell aggregates in computer-controlled suspension bioreactors.

Abstract: The clinical use of neural precursor cells (NPCs) for the treatment of neurological diseases, such as Parkinson's disease and Huntington's disease, requires overcoming the scarcity of these cells through controlled expansion. The main objective of the present study was to develop a large-scale computer-controlled bioprocess for the expansion of mammalian NPCs in suspension culture by scaling up existing reactor protocols. In order to support the oxygen demands of the maximum cell densities achieved, the volumetric mass transfer coefficient was kept above 1.10/h while scaling-up from small-scale 125 mL vessels to large-scale 500 mL bioreactors. In addition, the maximum shear stress at the impeller tip was maintained between 0.30 and 0.75 Pa to reduce damage to the cells. The resulting large-scale bioprocess achieved maximum viable cell densities of 1.2 x 10(6) cells/mL and a batch multiplication ratio of 9.1. Moreover, the process successfully maintained the NPC characteristics observed in small-scale studies.

Method and apparatus for analyzing bioprocess fluids

Abstract: Methods and apparatus for analyzing bioprocess fluids are provided A plurality of particles coated with a plurality of capture agents having an affinity for one or more biological markers is combined with bioprocess fluid to form a plurality of analyte-particle complexes The system also includes a transport arrangement for transporting the sample to a sensor surface, and optionally a magnetic field inducing structure constructed and arranged to establish a magnetic field at and adjacent to the sensor surface The resonant sensor produces a signal corresponding to an amount of analyte-particle complexes that are bound to the sensor surface

Enabling Technologies: Fermentation and Downstream Processing

Abstract: Efficient parallel tools for bioprocess design, consequent application of the concepts for metabolic process analysis as well as innovative downstream processing techniques are enabling technologies for new industrial bioprocesses from an engineering point of view. Basic principles, state-of-the-art techniques and cutting-edge technologies are briefly reviewed. Emphasis is on parallel bioreactors for bioprocess design, biochemical systems characterization and metabolic control analysis, as well as on preparative chromatography, affinity filtration and protein crystallization on a process scale.

High-throughput screening and process optimisation

Abstract: Introduction The biological production of active compounds, ranging from small molecules, such as organic acids, vitamins or antibiotics, through to macromolecules, such as therapeutic proteins or plasmid gene therapy vectors, is of great commercial and social value. The cornerstone of any such bioprocess is the cell cultivation step where a highly selected, and usually engineered, cell-line is grown under carefully controlled conditions. The term cell-line is used here to represent both microbial and mammalian cells. The aim of the cultivation step is to yield the product in as an efficient and cost-effective manner as possible. However, the design and implementation of a cell cultivation process is often a complex, lengthy and costly task. The development of a cell cultivation process typically involves four stages, as shown in Fig. 12.1. Stage 1 involves the initial identification of a native, or wild-type, cell-line that produces the compound of interest, though usually slowly and at low levels. This is followed by Stage 2 in which the productivity of the chosen cell-line [g product (g cells) −1 h −1 ] is enhanced using a variety of microbiological and molecular biology techniques. Stage 3 involves optimisation of the growth media composition and culture conditions; while in Stage 4 , scale-up of the process from laboratory, through pilot plant to manufacturing scale occurs. High-throughput experimentation Traditionally cell culture development has been carried out following a sequential series of experiments, using conventional apparatus with a high labour requirement. Conventional experimentation usually involves carrying out one or only a few experiments at a time.

System and Method for Bioprocess Control

Abstract: A system and method for controlling a bioprocess equipment (FIG. 1 ) includes developing a process model. The process model can be applied for process control purposes.

Monitoring of stress responses

Abstract: New developments in the RNA analysis techniques now enable a comprehensive view on the bacterial physiology under bioprocess conditions. The DNA-chip technology allows a genome wide transcriptional profiling of bacterial cells, whose genome sequence is available. Although the analyses of microbial bioprocesses have still been somewhat limited to date, this technique has already been successfully applied in different laboratories for the investigation of stress responses of selected industrially relevant bacterial hosts. Transcriptome analyses in combination with high resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry have been extensively applied for the description of general and specific stress and starvation responses of Escherichia coli and Bacillus subtilis. The consideration of bacterial stress and starvation responses is of crucial importance for the successful establishment of an industrial large scale bioprocess. Stress genes can be used as marker genes in order to monitor the fitness of industrial bacterial hosts during fermentation processes. This chapter gives an overview of current RNA analysis techniques. The bacterial stress and starvation responses, which are of potential importance for industrial microbial bioprocesses are summarised.

Biomass estimation using fluorescence measurements in Pichia pastoris bioprocess

Abstract: The utilisation of tryptophan fluorescence as an indirect biomass measurement for the yeast Pichia pastoris, an excellent host system for the production of heterologous proteins, is presented. Direct fluorescence measurements for cell densities above 3 g dm−3 presented important interferences due to inner filter effects. To overcome this drawback, a dilution protocol is provided which allows the quenching of the emission signal caused by solid particles to be controlled. The measured tryptophan fluorescence intensities were used to estimate biomass concentration during a P pastoris batch bioprocess growing either on glycerol or methanol. The best measurement model tested was based on the application of a Luedeking–Piret-based equation to fluorometric measurements. Thus, a linear relationship between the specific fluorescence evolution rate and specific growth rate was applied. The mean absolute relative prediction error (MARE) for biomass concentration was about 6%. Copyright © 2005 Society of Chemical Industry

Process analytical technology (PAT) needs and applications in the bioprocess industry

Abstract: Title Process analytical technology (PAT) needs and applications in the bioprocess industry Review Abstract Process analytical technologies have been applied to manufacturing processes (pulp and paper, chemical and petroleum) for decades. Recently, the US Food and Drug Administration (FDA) has, however, redefined the phrase and implemented it into an initiative focusing on improving several aspects of the pharmaceutical industry. The European Agency for the Evaluation of Medicinal Products (EMEA), has also been active and formed a PAT team in 2003. The PAT initiative was initially intended for traditional pharmaceutical manufacturers, but the FDA's PAT guidance now clearly states that it applies to all manufacturers of human and veterinary drug products, as well as biologics regulated by the FDA's Center for Drug Evaluation and Research (CDER) and the Center for Veterinary Medicine (CVM). Basically, PAT involves a fundamental shift from testing the quality of the finished drug product, to building quality into products by testing at several intermediate steps. It specifically requires that quantifiable, causal, and predictive relationships be established amongst raw materials, the manufacturing process, and final product quality. It is believed that PAT may not bring dramatic changes overnight, but years from now, it may be seen as an initiative that helped foster a period of innovation, efficiency, and expansion for the biopharmaceutical industry. In this report, the impact and potential effects of PAT on the biotechnological production of pharmaceuticals is assessed. Hence, we define BioPAT as process analytical technologies applied throughout development, scale-up and commercial scale bioprocess-based production of drug substances. In this report, we will focus on what PAT means in practice for the biotechnological manufacture of pharmaceuticals. Besides a regulatory examination, the monitoring methods and technologies available are thoroughly surveyed. These facts are then reflected against the needs for monitoring in bioprocess-based pharmaceutical production. Process analytical technologies have been applied to manufacturing processes (pulp and paper, chemicals and petroleum) for decades. Recently, the US Food and Drug Administration (FDA) has, however, redefined the phrase and implemented it into an initiative focusing on improving several aspects of the pharmaceutical industry. The first document in support of this action, the process analytical technology (PAT) initiative, was launched by the FDA in 2002, followed up by the PAT guidance in September, 2004. The FDA's European counterpart, the European Agency for the Evaluation of Medicinal Products (EMEA), has also been active and formed a PAT team in 2003. The PAT …

Horizontal tubular bioreactors in biotechnology

Abstract: In this review, horizontal tubular bioreactors are discussed regarding their advantages and disadvantages compared to other bioreactor types. In horizontal tubular bioreactors medium flow is characterized by plug flow conditions that can be favorable in the case of inhibition and/or repression bioprocess kinetics. For description of liquid flow simple one-parameter or complex multi-parameters mathematical models have been established. Comparison between these models proved that complex multi-parameters model can describe real situation in the bioreactor more efficiently. Criteria of geometrical similarity are most often used for scale-up of horizontal tubular bioreactors. The successful scale-up procedure should combine the mathematical model of medium flow behavior with bioprocess kinetics in the bioreactor.

Screening and Industrial Application of Unique Microbial Reactions Involved in Nucleic Acid and Lipid Metabolisms

Abstract: Bioprocesses, which involve biocatalysts for the production of useful compounds, are expected to become a leading player in green chemistry. The first step in bioprocess development is screening for useful biological reactions in the immense number of microorganisms with infinite diversity and versatility. This review introduces some examples of bioprocess development that started from process design stemming from the discovery of unique metabolic processes, reactions, and enzymes in microbial nucleic acid and lipid metabolisms.

Development of biotechnological processes by integrating genetic and engineering methods

Abstract: The production systems and product lines used in the collaborative research centre (SFB 578) are considered to be an example for the superior goal to develop new tools, methods, data, and models for bioprocess design. Integrating genetic, biologic, biochemical and engineering methods for process design is helpful to reduce the development efforts and costs. Basic parameters describing the whole process have to be found and systems biotechnological models have to be developed. They should enable to synthesize products in biologically active form in high concentration, yield, and purity with as few production steps as possible.

An integrated anaerobic/aerobic bioprocess for the remediation of chlorinated phenol-contaminated soil and groundwater.

Abstract: An investigation of biodegradation of chlorinated phenol in an anaerobic/aerobic bioprocess environment was made. The reactor configuration used consisted of linked anaerobic and aerobic reactors, which served as a model for a proposed bioremediation strategy. The proposed strategy was studied in two reactors before linkage. In the anaerobic compartment, the transformation of the model contaminant, 2,4,6-trichlorophenol (2,4,6-TCP), to lesser-chlorinated metabolites was shown to occur during reductive dechlorination under sulfate-reducing conditions. The consortium was also shown to desorb and mobilize 2,4,6-TCP in soils. This was followed, in the aerobic compartment, by biodegradation of the pollutant and metabolites, 2,4-dichlorophenol, 4-chlorophenol, and phenol, by immobilized white-rot fungi. The integrated process achieved elimination of the compound by more than 99% through fungal degradation of metabolites produced in the dechlorination stage. pH correction to the anaerobic reactor was found to be necessary because acidic effluent from the fungal reactor inhibited sulfate reduction and dechlorination.

Development of an Efficient Bioprocess for Poultry Vaccines Using High‐density Insect Cell Culturea

Abstract: Infectious bursa1 disease virus (IBDV) is a pathogen of major economic importance to the world’s poultry industries. It causes severe immunodeficiency in young chickens by destroying the precursors of antibody-producing B cells in the bursa of Fabricius.’ IBDV is a member of the Birnaviridae family whose genome consists of two segments of double-stranded RNA. Presently, the principle method of controlling IBDV infection in young chickens is by vaccination with an avirulent strain of IBDV or by the transfer of high levels of maternal antibody to breeder hens.* Recently, virulent strains that are antigentically different from previously established IBDVs have been isolated from vaccinated flocks on the Delmarva peninsula. Consequently, present vaccines afford only partial protection against infection. In FIGURE 1,the large segment indicated is comprised of a precursor polyprotein that is processed into mature VP2, VP3, and VP4.3 VP2 and VP3 are the major structural proteins of the virion. VP2 is the major host-protective immunogen of IBDV and contains the antigenic regions responsible for the induction of neutralizing antibodies. For this reason, we have focused on VP2 as a potential vaccine. When expressed in baculovirus as one of a cassette (VP2, VP3, and VP4), the coat proteins self-assemble into empty virions, which subsequently afford protection in challenged chickens (FIG. 2): This article presents the current status of our efforts to develop a cost-efficient vaccine for IBDV using the recombinant baculovirus and insect cell culture process. Although the baculovirus expression system has proven quite successful for use in laboratories, its use as an industrial expression system has yet to occur, although products for human use are in clinical trials. Briefly, the system entails replacing the gene for the AcNPV coat protein, polyhedrin, with the gene for the protein of