Process modeling and simulation can guide process development: case study α-cyclodextrin

Modeling and simulation of biotechnological processes enables the quantification of possible process variations. In this way it provides a tool for guiding research towards the most promising directions. The method is shown on the basis of the case study of α-cyclodextrin production. The production process was modeled and the model is used to evaluate the potential impact of CGTases with new properties. Sensitivity analyses show that yield and selectivity are the crucial parameters. Reaction time has only limited impact and substrate concentration has to be above 20%. The use of thermostable enzymes provides some energy savings. Furthermore, the effect of new CGTases leading to a modified downstream processing was assessed.     

Separation of lactobacilli bacteriocins from fermented broths using membranes

Current separation, isolation and purification techniques to obtain highly potent purified lactobacilli and lactococci bacteriocins include chemical precipitation, separation employing solvents and chromatographic techniques. These methods are arduous, costly, with limited scalability, offering low bacteriocin yields (<20%). To address these challenges, the alternatives of ultrafiltration and nanofiltration, as separation methods were tested. Three promising bacteriocin producing strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and Lactococcus lactis NCIMB 8586 were selected to investigate the applicability and feasibility of the method.To facilitate separation, the microorganisms were grown on specially developed low molecular weight medium (LMWM) mainly containing nutritive sources up to 4 kDa molecular weight. Bacterial cells were removed by centrifugation. The clarified broths were filtered using 4 and 1 kDa MWCO. Bacteriocin activity was determined by an antimicrobial activity test using nisin, which has an inhibitory effect on the growth of susceptible microorganisms. Recovery yields using filtration were found to range between 53 and 68%, a high recovery performance.The bacteriocin activity of crude extracts of all the three lactobacilli were between 95 and 105 IU ml^?1. When the substances were separated using ultrafiltration membrane (4 kDa MWCO) their activity was enhanced to 145–150 IU ml^?1, achieving a total potency yield of 44–53%. Further enhancement of yields up to 36% was attained employing nanofiltration (1 kDa MWCO) membranes with an activity increased up to 200 IU ml^?1.Bacteriocin isolation from crude extracts using filtration was found to be effective, offering high recovery yields, optimising their activity as well as presenting a realistic option towards the formulation of these as commercially available antibacterial agents.     

Involve both genetic and environmental factors to build monkey models of mental disorders

正Dear Editor,Mental diseases,such as schizophrenia,are typically the result of multiple abnormalities,including neurobiological,psychological and sociological processes,particularly the environmental and genetic disorders(Bailey,2000).Consequently,in psychic medicine,animal models should be developed via methods consistent with their associated theories of mental disorders.However,many conventional models are constructed via genetic manipulations or surgical operations to the nervous system(Bezard,2006,van der Staay,2006,2009),e.g.administrate animals with agonists or antagonists of various neurotransmitters or drugs to reproduce human mental disorders.The usage of these methods,which should be considered as simple neuro-pharmacological     

Negative chromatography: Progress,applications and future perspectives

In negative chromatography, the impurities bind on the adsorbent, and the product is allowed to flow through the chromatographic column. Negative chromatography is an alternative to positive chromatography under certain circumstances and has been used to purify various biomolecules. For this review, a detailed survey of the performance of reported studies on negative chromatography was conducted. The applications of negative chromatography in the capture and intermediate purification steps for biomolecules (e.g., plasmid DNA, antibodies, enzymes, hemoglobin, virus particles and cells) are reviewed. The negative chromatographic adsorbents adsorb the impurities through surface charge, hydrophobic interaction at specific sites on the surface, hydrophobic interaction, hydrogen bonding and functional groups. Examples of applications of negative chromatography according to the type of chromatography matrix used are summarized and discussed. In addition, the effects of operating conditions (initial protein concentration, buffer ions, pH and salt concentration) are discussed, and the criteria for choosing negative or positive chromatography are summarized. The literature survey showed that there will be future limitations and challenges ahead in implementation of negative chromatography. Possible solutions to the limitations and challenges of negative chromatography and future trends for developing negative chromatography are discussed.     

On the stability of plasmid DNA vectors during cell culture and purification

Gene therapy and DNA vaccination applications have increased the demand for highly purified plasmid DNA (pDNA) in the last years. One of the main problems related to the scale-up of pDNA purification is the degradation of the supercoiled (sc) isoforms during cell culture and multi-stage purification. In this work, a systematic study of the stability of two model plasmids (3,697 and 6,050 bp) during a mid-scale production process, which includes fermentation, alkaline lysis, isopropanol and ammonium sulphate precipitation and hydrophobic interaction chromatography, was performed. Results indicate that by extending cell culture (up to 26 h) and cell lysis (up to 2 h) it is possible to significantly reduce the amounts of RNA, without significantly compromising the yields of the sc pDNA isoform, a feature that could be conveniently exploited for downstream processing purposes. The stability of pDNA upon storage of E. coli pellets at different temperatures indicates that, differently from RNA, pDNA is remarkably stable when stored in cell pellets (>3 weeks at 4°C, >12 weeks at −20°C) prior to processing. With alkaline lysates, however, storage at −20°C is mandatory to avoid sc pDNA degradation within the first 8 weeks. Furthermore, the subsequent purification steps could be carried out at room temperature without significant pDNA degradation. Since the unit operations and process conditions studied in this work are similar to those generally used for plasmid DNA production, the results presented here may contribute to improve the current knowledge on plasmid stability and process optimization. Authors Freitas and Azzoni contributed equally to this work.     

Combination of biotransformation and chromatography for the isolation and purification of mannoheptulose

Mannoheptulose is a seven-carbon sugar. It is an inhibitor of glucose-induced insulin secretion due to its ability to selectively inhibit the enzyme glucokinase. An improved procedure for mannoheptulose isolation from avocados is described in this study (based upon the original method by La Forge). The study focuses on the combination of biotransformation and downstream processing (preparative chromatography) as an efficient method to produce a pure extract of mannoheptulose. The experiments were divided into two major phases. In the first phase, several methods and parameters were compared to optimize the mannoheptulose extraction with respect to efficiency and purity. In the second phase, a mass balance of mannoheptulose over the whole extraction process was undertaken to estimate the yield and efficiency of the total extraction process. The combination of biotransformation and preparative chromatography allowed the production of a pure mannoheptulose extract. In a biological test, the sugar inhibited the glucokinase enzyme activity efficiently.     

A massively asynchronous,parallel brain

Whether the visual brain uses a parallel or a serial, hierarchical, strategy to process visual signals, the end result appears to be that different attributes of the visual scene are perceived asynchronously—with colour leading form (orientation) by 40 ms and direction of motion by about 80 ms. Whatever the neural root of this asynchrony, it creates a problem that has not been properly addressed, namely how visual attributes that are perceived asynchronously over brief time windows after stimulus onset are bound together in the longer term to give us a unified experience of the visual world, in which all attributes are apparently seen in perfect registration. In this review, I suggest that there is no central neural clock in the (visual) brain that synchronizes the activity of different processing systems. More likely, activity in each of the parallel processing-perceptual systems of the visual brain is reset independently, making of the brain a massively asynchronous organ, just like the new generation of more efficient computers promise to be. Given the asynchronous operations of the brain, it is likely that the results of activities in the different processing-perceptual systems are not bound by physiological interactions between cells in the specialized visual areas, but post-perceptually, outside the visual brain.     

Data Cubes and Matrix Formulae for Convenient Handling of Physical Flow Data

If the technosphere and the biosphere are divided into cells, the presence and turnover of a substance in a study area can be summarized in a vector of stocks and a matrix of flows between different pairs of cells. Likewise the stocks and flows of several substances or materials in one or more time periods can be summarized in multidimensional data cubes. In this article, we provide a theoretical framework for handling physical flow data, and we demonstrate how a set of matrix operations can facilitate exploratory analysis and quality assessment of such data regardless of the number of substances, materials, and time periods considered. In particular, we show how matrices and cubes of flow data can be recalculated when the collection of cells is modified by joining cells, and also what information is required to recalculate flows when cells are split. Furthermore, we illustrate how and under what circumstances substance-flow data originating from different studies with different collections of cells can be compared or merged. The generic character of the given formulae facilitates the development of software for physical flow data.     

Upstream and downstream strategies to economize biodiesel production

In recent years biodiesel has drawn considerable amount of attention as a clean and renewable fuel. Biodiesel is produced from renewable sources such as vegetable oils and animal fat mainly through catalytic or non-catalytic transesterification method as well as supercritical method. However, as a consequence of disadvantages of these methods, the production cost increases dramatically. This article summarizes different biodiesel production methods with a focus on their advantages and disadvantages. The downstream and upstream strategies such as using waste cooking oils, application of non-edible plant oils, plant genetic engineering, using membrane separation technology for biodiesel production, separation and purification, application of crude glycerin as an energy supplement for ruminants, glycerin ultra-purification and their consequent roles in economizing the production process are fully discussed in this article.     

A single-use purification process for the production of a monoclonal antibody produced in a PER.C6 human cell line

Advances in single-use technologies can enable greater speed, flexibility, and a smaller footprint for multi-product production facilities, such as at a contract manufacturer. Recent efforts in the area of cell line and media optimization have resulted in bioreactor productivities that exceed 8 g/L in fed-batch processes or 25 g/L in high-density cell culture processes. In combination with the development of single-use stirred tank bioreactors with larger working volumes, these intensified upstream processes can now be fit into a single-use manufacturing setting. Contrary to these upstream advances, downstream single-use technologies have been slower to follow, mostly limited by low capacity, high cost, and poor scalability. In this study we describe a downstream process based solely on single-use technologies that meets the challenges posed by expression of a mAb (IgG(1)) in a high-density suspension culture of PER.C6 cells. The cell culture harvest was clarified by enhanced cell settling (ECS) and depth filtration. Precipitation was used for crude purification of the mAb. A high capacity chromatographic membrane was then used in bind/elute mode, followed by two membranes in flow-through (FT) mode for polishing. A proof of concept of the entire disposable process was completed for two different scales of the purification train.