Showing papers on "Bioprocess published in 2005"

Differentiation and lineage selection of mouse embryonic stem cells in a stirred bench scale bioreactor with automated process control

Abstract: It is well established that embryonic stem (ES) cells can differentiate into functional cardiomyocytes in vitro. ES-derived cardiomyocytes could be used for pharmaceutical and therapeutic applications, provided that they can be generated in sufficient quantity and with sufficient purity. To enable large-scale culture of ES-derived cells, we have developed a robust and scalable bioprocess that allows direct embryoid body (EB) formation in a fully controlled, stirred 2 L bioreactor following inoculation with a single cell suspension of mouse ES cells. Utilizing a pitched-blade-turbine, parameters for optimal cell expansion as well as efficient ES cell differentiation were established. Optimization of stirring conditions resulted in the generation of high-density suspension cultures containing 12.5 × 106 cells/mL after 9 days of differentiation. Approximately 30%–40% of the EBs formed in this process vigorously contracted, indicating robust cardiomyogenic induction. An ES cell clone carrying a recombinant DNA molecule comprised of the cardiomyocyte-restricted alpha myosin heavy chain (αMHC) promoter and a neomycin resistance gene was used to establish the utility of this bioprocess to efficiently generate ES-derived cardiomyocytes. The genetically engineered ES cells were cultured directly in the stirred bioreactor for 9 days, followed by antibiotic treatment for another 9 days. The protocol resulted in the generation of essentially pure cardiomyocyte cultures, with a total yield of 1.28 × 109 cells in a single 2 L bioreactor run. This study thus provides an important step towards the large-scale generation of ES-derived cells for therapeutic and industrial applications. © 2005 Wiley Periodicals, Inc.

Development of a perfusion fed bioreactor for embryonic stem cell-derived cardiomyocyte generation: oxygen-mediated enhancement of cardiomyocyte output.

Abstract: Cell transplantation is emerging as a promising new approach to replace scarred, nonfunctional myocardium in a diseased heart. At present, however, generating the numbers of donor cardiomyocytes required to develop and test animal models is a major limitation. Embryonic stem (ES) cells may be a promising source for therapeutic applications, potentially providing sufficient numbers of functionally relevant cells for transplantation into a variety of organs. We developed a single-step bioprocess for ES cell-derived cardiomyocyte production that enables both medium perfusion and direct monitoring and control of dissolved oxygen. Implementation of the bioprocess required combining methods to prevent ES cell aggregation (hydrogel encapsulation) and to purify for cardiomyocytes from the heterogeneous cell populations (genetic selection), with medium perfusion in a controlled bioreactor environment. We used this bioprocess to investigate the effects of oxygen on cardiomyocyte generation. Parallel vessels (250 mL culture volume) were run under normoxic (20% oxygen tension) or hypoxic (4% oxygen tension) conditions. After 14 days of differentiation (including 5 days of selection), the cardiomyocyte yield per input ES cell achieved in hypoxic vessels was 3.77 +/- 0.13, higher than has previously been reported. We have developed a bioprocess that improves the efficiency of ES cell-derived cardiomyocyte production, and allows the investigation of bioprocess parameters on ES cell-derived cardiomyogenesis. Using this system we have demonstrated that medium oxygen tension is a culture parameter that can be manipulated to improve cardiomyocyte yield.

Parallel reactor systems for bioprocess development.

Abstract: Controlled parallel bioreactor systems allow fed-batch operation at early stages of process development. The characteristics of shaken bioreactors operated in parallel (shake flask, microtiter plate), sparged bioreactors (small-scale bubble column) and stirred bioreactors (stirred-tank, stirred column) are briefly summarized. Parallel fed-batch operation is achieved with an intermittent feeding and pH-control system for up to 16 bioreactors operated in parallel on a scale of 100 ml. Examples of the scale-up and scale-down of pH-controlled microbial fed-batch processes demonstrate that controlled parallel reactor systems can result in more effective bioprocess development. Future developments are also outlined, including units of 48 parallel stirred-tank reactors with individual pH- and pO2-controls and automation as well as liquid handling system, operated on a scale of ml.

Applications of metabolic modeling to drive bioprocess development for the production of value-added chemicals

Abstract: Increasing numbers of value added chemicals are being produced using microbial fermentation strategies. Computational modeling and simulation of microbial metabolism is rapidly becoming an enabling technology that is driving a new paradigm to accelerate the bioprocess development cycle. In particular, constraint-based modeling and the development of genome-scale models of industrial microbes are finding increasing utility across many phases of the bioprocess development workflow. Herein, we review and discuss the requirements and trends in the industrial application of this technology as we build toward integrated computational/experimental platforms for bioprocess engineering. Specifically we cover the following topics: (1) genome-scale models as genetically and biochemically consistent representations of metabolic networks; (2) the ability of these models to predict, assess, and interpret metabolic physiology and flux states of metabolism; (3) the model-guided integrative analysis of high throughput ‘omics’ data; (4) the reconciliation and analysis of on- and off-line fermentation data as well as flux tracing data; (5) model-aided strain design strategies and the integration of calculated biotransformation routes; and (6) control and optimization of the fermentation processes. Collectively, constraint-based modeling strategies are impacting the iterative characterization of metabolic flux states throughout the bioprocess development cycle, while also driving metabolic engineering strategies and fermentation optimization.

Process simulation and modeling for industrial bioprocessing: Tools and techniques

Abstract: Maximizing profits by operating the most efficient process is the primary goal of all industrial bioprocessing operations. To help create efficient operations companies use process simulation, which is the application of a range of software tools to analyze complete processes, not just single unit operations. Process engineers and scientists use simulation models to investigate complex and integrated biochemical operations, without the need for extensive experimentation.

Use of operating windows in the assessment of integrated robotic systems for the measurement of bioprocess kinetics.

Abstract: This study examines the utility of an automated liquid handling robot integrated with a microwell plate reader to enable the rapid acquisition of bioprocess kinetic data. The relationship between the key parameters for liquid handling accuracy and precision and the sample detection period has been characterized for typical low-viscosity (<2.0 mPa x s) aqueous and organic phases and for a high-viscosity aqueous phase (60 mPa x s), all exhibiting Newtonian rheology. The use of a simple graphical method enables the suitability of a given automation platform to be assessed once the user has determined the minimum sample detection period and the minimum accurate and precise dispense volume. This provides for a reduction in the duration of any experiment by maximizing well usage within each microwell plate. The suitability of employing an integrated automation platform to gather kinetic data for systems typical of those encountered in bioprocessing is analyzed via a series of case studies. Application to alkaline cell lysis, where disruption is complete within 120 s, showed that the range of available dispense volumes and the number of wells that can be utilized is limited. In contrast, analysis of a system exhibiting slow process kinetics, the fermentation of Escherichia coli TOP10 pQR239 in microwell plates, demonstrated that, for a typical sample detection period of 30 min, the only restrictions on the degree of well utilization are the liquid handling accuracy and precision and the volume capacity of the liquid handling robot. Finally, liquid-liquid extraction, an example of a kinetically independent operation, was also examined. In this case, only a single equilibrium measurement is required, which means that the only restrictions to the utilization of the integrated devices are the liquid handling accuracy and precision. Integrated automation platforms represent a powerful process development tool over traditional experimental methods used for bioprocess development. Smaller volumes of reagent and sample can be used to achieve greater throughput, while high levels of reproducibility and sensitivity are maintained.

Downstream processing in marine biotechnology.

Abstract: Downstream processing is one of the most underestimated steps in bioprocesses and this is not only the case in marine biotechnology. However, it is well known, especially in the pharmaceutical industry, that downstreaming is the most expensive and unfortunately the most ineffective part of a bioprocess. Thus, one might assume that new developments are widely described in the literature. Unfortunately this is not the case. Only a few working groups focus on new and more effective procedures to separate products from marine organisms. A major characteristic of marine biotechnology is the wide variety of products. Due to this variety a broad spectrum of separation techniques must be applied. In this chapter we will give an overview of existing general techniques for downstream processing which are suitable for marine bioprocesses, with some examples focussing on special products such as proteins (enzymes), polysaccharides, polyunsaturated fatty acids and other low molecular weight products. The application of a new membrane adsorber is described as well as the use of solvent extraction in marine biotechnology.

Large Scale Bioprocess for the Production of Optically Pure L-Carnitine

Abstract: A competitive production method using the biotransformation of 4-butyrobetaine to enantiomerically pure L-carnitine was developed and scaled-up by Lonza The process produces L-carnitine in 995% yield, and >999% enantiomeric excess (ee) Continuous and discontinuous processes were developed but the fed-batch process was found to be economically the most favourable process mode

Biooxidation capabilities of Candida sp

Abstract: A bioprocess for producing carboxylic acids, alcohols and aldehydes is provided by culturing Candida sp. in a fermentation medium containing various defined substrates.

Chapter 11 Biosensors for bioprocess monitoring

Abstract: Publisher Summary This chapter focuses on the biosensors for bioprocess monitoring. Biosensor development for bioprocess monitoring focused on the development of reliable and stable automated systems for the determination of nutrients (e.g., glucose) and metabolic products (e.g., lactate, ethanol, and glutamine). The chapter discusses approaches for on-line monitoring and control of substrate and product concentrations with enzyme electrodes. The chapter focuses on the contributions of biosensors for the determination of product quality and gene copy number. It also explores the biosensors for the quantification of plasmid DNA and the biological activity of proteins. It also discusses enzyme-based on-line analysis of indicator compounds for cell physiology or metabolic stress and arrays for gene expression analysis.

Simultaneous wastewater nutrient removal by a novel hybrid bioprocess

Abstract: A combined activated sludge–biofilm bioprocess called TNCU-I was developed by adding a rotating biological contactor to the aerobic zone of a traditional A2O process in order to solve the sludge retention time conflict between nitrifiers and phosphate accumulating organisms (PAOs), and the carbon source competition between denitrifiers and PAO. The TNCU-I process shows excellent carbon, nitrogen, and phosphate removal performance when treating synthetic wastewater. The process also achieved a more stable nitrification performance than the A2O process. The specific nitrification rate, the specific anoxic and aerobic phosphate uptake rates, the specific denitrification rate, and the specific anaerobic phosphate release rate were determined by a series of batch experiments. Such data were further analyzed to optimize the volume ratio of the TNCU-I anaerobic, anoxic, and aerobic tanks. The optimized process was also operated to confirm the performance. In addition, both Nitrosospira and Nitrospira were identi...

Research progress of converting lignocellulose to produce fuel ethanol

Abstract: The conversion process of ethanol fuel utilizing lignocelluloses as a substrate involves pretreatment,enzymatic hydrolysis and fermentation.The new technological progress and the methods of solving present problems in these processes are extensively reviewed.Ammonia fiber explosion (AFEX) process is an attractive pretreatment method,moreover,ultrasonic and microwave treatments contribute to enhancing enzymatic hydrolysis.The enzymatic hydrolysis mechanism for cellulose,the production of cellulase and the optimization methods for enzymatic hydrolytic process are provided.Consolidated bioprocess technology is promising in lignocellulose biological conversion,furthermore,selecting suitable means of fermentation,optimizing fermentative process and solving inhibitor problem during the fermentative processes are significant to higher ethanol yields.Genetic engineering of bacteria to ferment the diverse range of sugar is a milestone essential to the bioethanol production.

On the stability of a linear bioprocess model with recycle loop

Abstract: In this note, we treat a linear bioprocess model with recycle loop and analyze the spectrum of the closed-loop operator in order to discuss the exponential stability. It is shown that a C/sub 0/-semigroup generated by the closed-loop operator satisfies the spectrum determined growth assumption and that the recycle loop works effectively for the process through a numerical simulation.

Using the complex-step derivative approximation method to calculate local sensitivity functions of highly nonlinear bioprocess models

Abstract: In this paper, the complex-step derivative approximation technique will be used for calculating local sensitivity functions. This technique is compared to the finite difference approximation, probably the most used local sensitivity analysis technique. For this comparison, 4 biotechnological models with varying model complexity were used. A well known problem of the finite difference approximation is the choice of a suitable perturbation factor in order to avoid non-linear model effects or numerical errors due to the subtraction of almost equal numbers. The main advantage of the complex-step derivative approximation technique is that it is not susceptible to errors introduced by small perturbation factors, ruling out the entire search for optimal perturbation factors. However, the main disadvantage is an important execution time increase for large models. Keywords—Local sensitivity analysis, complex-step, finite difference, numerical methods, biotechnology.

Transient Transfection of HEK293 Cells in Suspension : Process Characterization and Optimization by Applying Invasive Nucleotide and Non-invasive Electronic Nose Technology

Abstract: The increasing demand for milligrams of recombinant proteins (r-proteins) to be used for therapeutics and structural studies justifies the need for a rapid and scalable expression system. Transient transfection of mammalian cells is a powerful technology for the fast production of large amounts of these r-proteins. This system depends on a multitude of different factors including the cell line, its physiological state, the type of expression vector and the medium formulation. Moreover, monitoring bioprocess parameters, and control of the bioprocess at its optimal state, enable the reduction of production costs, increase the yield and maintain the quality of the desired product. Off-line and on-line monitoring methods have been used to characterize and to optimize the transient transfection process using HEK293 cells and polyethylenimine as transfection mediator. As an off-line method the intracellular nucleotide pool was used, which has been considered as a reliable tool reflecting the metabolic status, the growth potential, and the overall physiological condition of the cell. As an on-line and non-invasive method a completely new technology, the electronic nose, was applied for bioprocess monitoring and the identification of characteristic process states. HEK293 cells were cultured in suspension (HEK293s) using a completely new medium, totally free of proteins or animal-origin components, which facilitates the transfection process by allowing growth and transfection of the cells without medium exchange. This is a central pre-requisite for scaling the technology up to large volume cultivation processes. A special bicistronic plasmid was constructed under control of the cytomegalovirus promoter. The plasmid DNA was combined with polyethylenimine (PEI) using an efficient minimum amount of DNA at a DNA:PEI ratio of 0.50:1.50 (µg:µg) showing 70-80 % of GFP positive cells under serum-containing as well as serum-free culture conditions. Specific cells taken from an early (from 40 to 77) and a late (from 89 to 150) passage number were compared as hosts for transient transfection. The combined NucleotideTriPhosphate ratio expressed as NTP/U = [ATP+GTP] [UDP-GNAc] / [UTP+CTP] [UTP] was calculated in parallel during cultivation and transient transfection under serum-containing and serum-free culture conditions for process monitoring and characterization. The results showed that nucleotide ratios can be used to distinguish between: HEK293s cells from early and late passage numbers; Cells cultured under serum-containing conditions from those under protein/serum-free medium conditions; Transfected and untransfected cells in culture; DNA:PEI complex uptake (transfection procedure) from PEI uptake alone. The BioNose was connected to the off-gas line of the bioreactor cultivation. The data were collected using the NST Senstool software and evaluated applying multivariate methods such as Principal Component Analysis (PCA). The results showed, that the BioNose can generate characteristic pattern specific for a particular process being an easily accessible tool to monitor cell cultivations as well as to distinguish between different culture conditions.

State Reconstruction in Spatially Distributed BioProcess Systems using Reduced Order Models: Application to the Gluconic Acid Production.

Abstract: In this work, the dissipative nature of spatially distributed bioprocess systems is exploited to develop efficient state observers based on a low dimensional dynamic representation of the original set of partial differential equations. The approach we suggest combines standard observer design techniques for bioreactors with efficient model reduction methodologies based on projection of the original concentration fields on low dimensional subspaces capturing the slow dynamics of the process. Aspects related with the location of sensors and their influence on the ability to reconstruct concentration fields will also be considered. Finally, the different aspects of the methodology, as well as the efficiency of the resulting observers will be illustrated on a case study of industrial interest, namely a tubular bioreactor producing gluconic acid by Aspergillus Niger.

Modelling and identification of an activated sludge depollution bioprocess

Abstract: In this work the problem of modelling and identification of an activated sludge depollution bioprocess is focused. This bioprocess is in fact an aerobic fermentation process that is carried out in a recycle bioreactor. A nonlinear dynamical model is obtained using the reaction scheme and the mass balance. The dynamical kinetics of the process are strongly nonlinear and not exactly known; therefore an estimation strategy is developed for identification. The nonlinear observer design is based on high gain approach. The tuning of the observers is reduced to the calibration of a single parameter. Computer simulations are included.

Estrategias de bioingenieria para la recuperacion primaria de productos biologicos bioengineering strategies for the primary recovery of biological products

Abstract: The increasing interest of the pharmaceutical companies to develop efficient and scale-up processes, that allow them to rapidly bring new products to the market, has forced them to develop new bioengineering strategies. One of the current trend is to exploit bioprocess integration and intensification approaches for the development of recovery and purification processes for biological products, particularly proteins. This article presents some cases of the results of the practical application of these approaches, using the techniques of aqueous two phase system and expanded bed