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Downstream Processing (downstream + processing)
Selected AbstractsCrystallization for the Downstream Processing of ProteinsENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2005S. Schmidt Abstract Protein crystallization offers great potential in downstream processing of pharmaceutical protein active ingredients. The advantages, which are well known and widely utilized in low-molecular weight crystallization, can also be expected to be found to some extent in protein crystallization. However, there is still a marked need for improvement in two main areas of protein processing, namely, in crystallization from impure solutions and scale-up. [source] Emergence of Ideal Membrane Cascades for Downstream ProcessingBIOTECHNOLOGY PROGRESS, Issue 3 2008Edwin N. Lightfoot An algorithm is developed for describing ideal membrane cascades for fractionation of binary and pseudo-binary mixtures. It is shown that solvent management plays a key role in determining both purification and yield. Development of efficient diafilters is needed if membrane cascades are to achieve their full potential in competing with both chromatography and simulated moving bed operations in downstream processing of proteins. Such a replacement will also be important for fractionation of higher titers and larger substrates, such as plasmids, viruses, and even whole cells. [source] Applying Near-Infrared Spectroscopy in Downstream Processing: One Calibration for Multiple Clarification Processes of Fermentation MediaBIOTECHNOLOGY PROGRESS, Issue 2 2008Licínia O. Rodrigues The use of near-infrared spectroscopy (NIRS) is demonstrated in the first downstream processing (DSP) steps of an active pharmaceutical ingredient (API) manufacturing process. The first method developed was designed to assess the API content in the filtrate stream (aqueous) of a rotary drum vacuum filter. The PLS method, built after spectral preprocessing and variable selection, had an accuracy of 0.01% (w/w) for an API operational range between 0.20 and 0.45% (w/w). The robustness and extrapolation ability of the calibration was proved when samples from ultrafiltration and nanofiltration processes, ranging from 0 to 2% (w/w), were linearly predicted ( R2=0.99). The development of a robust calibration model is generally a very time-consuming task, and once established it is imperative that it can be useful for a long period of time. This work demonstrates that NIR procedures, when carefully developed, can be used in different process conditions and even in different process steps of similar unit operations. [source] Fluidized Bed Design Parameters Affecting Novel Lactic Acid Downstream ProcessingBIOTECHNOLOGY PROGRESS, Issue 6 2001Ana V. Sosa Lactic acid purification was directly done from fermentation utilizing a fluidized bed column refilled with a strong anionic exchange resin. The purpose of this work was to study the influence of two important design parameters, bed-diameter (D) and bed-height (H), in the lactic acid binding and elution capacity of the matrix. By changing the settled bed height from 2.5 to 5 cm for each diameter of column analyzed it was possible to obtain an 50% increase in the binding capacity of the resin in all experiments. This fact was attributed to a higher contact time between the culture broth and the anionic resin produced by the increase of back mixing and lactic acid residence time. [source] Downstream Processing of Enzymatically Produced Geranyl GlucosideBIOTECHNOLOGY PROGRESS, Issue 5 2001B. Mattheus de Roode Geraniol plays an important role in the fragrance and flavor industry. The corresponding glucoside has interesting properties as a "slow release" aroma compound. Therefore, the enzymatic production and downstream processing of geranyl glucoside were investigated. Geranyl glucoside was produced in a spray column reactor with an initial production rate of 0.58 mg U,1 h,1. A pretreated hydrophobic microfiltration membrane was used to prevent migration of the aqueous, enzyme-containing phase to the downstream process. No retention of the glucoside, which accumulated in the geraniol phase, was found. On the basis of examples from the literature, four downstream processes were tested on their viability for this system. Extraction with water and foaming were not suitable to recover geranyl glucoside from geraniol. In the first case, the glucoside selectivity for the geraniol phase was found to be high, which made extraction with water unsuccessful. In the second case it was possible to obtain a stable foam, but significant enrichment of the foam with glucoside did not occur. Adsorption on alumina and distillation under reduced pressure were applied successfully and tested in-line with the bioreactor. A maximum glucoside adsorption of 7.86 mg g,1 was achieved on alumina. After desorption and evaporation of the extractant the pure glucoside was obtained quantitatively. A pure product could not be obtained after distillation because a small amount of glucose was present in the permeate as well, which accumulated in the bottom fraction. It was shown that with this reactor system a production of 1 kg of geranyl glucoside in 2 days is possible using an initial amount of 50,000 units of enzyme. [source] Sulfated membrane adsorbers for economic pseudo-affinity capture of influenza virus particlesBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2009Lars Opitz Abstract Strategies to control outbreaks of influenza, a contagious respiratory tract disease, are focused mainly on prophylactic vaccinations in conjunction with antiviral medications. Currently, several mammalian cell culture-based influenza vaccine production processes are being established, such as the technologies introduced by Novartis Behring (Optaflu®) or Baxter International Inc. (Celvapan). Downstream processing of influenza virus vaccines from cell culture supernatant can be performed by adsorbing virions onto sulfated column chromatography beads, such as Cellufine® sulfate. This study focused on the development of a sulfated cellulose membrane (SCM) chromatography unit operation to capture cell culture-derived influenza viruses. The advantages of the novel method were demonstrated for the Madin Darby canine kidney (MDCK) cell-derived influenza virus A/Puerto Rico/8/34 (H1N1). Furthermore, the SCM-adsorbers were compared directly to column-based Cellufine® sulfate and commercially available cation-exchange membrane adsorbers. Sulfated cellulose membrane adsorbers showed high viral product recoveries. In addition, the SCM-capture step resulted in a higher reduction of dsDNA compared to the tested cation-exchange membrane adsorbers. The productivity of the SCM-based unit operation could be significantly improved by a 30-fold increase in volumetric flow rate during adsorption compared to the bead-based capture method. The higher flow rate even further reduced the level of contaminating dsDNA by about twofold. The reproducibility and general applicability of the developed unit operation were demonstrated for two further MDCK cell-derived influenza virus strains: A/Wisconsin/67/2005 (H3N2) and B/Malaysia/2506/2004. Overall, SCM-adsorbers represent a powerful and economically favorable alternative for influenza virus capture over conventional methods using Cellufine® sulfate. Biotechnol. Bioeng. 2009;103: 1144,1154. © 2009 Wiley Periodicals, Inc. [source] Highlights in Biocatalysis , Historical Landmarks and Current TrendsENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2005T. Bornscheuer Abstract Biocatalysis has ancient roots, yet it is developing into a key tool for synthesis in a wide range of applications. Important events in the history of enzyme technology from the 19th century onwards are highlighted. Considering the most relevant progress steps, the production of penicillanic acid and the impact of genetic engineering are traced in more detail. Applied biocatalysis has been defined as the application of a biocatalyst to achieve a desired conversion selectively, under controlled, mild conditions in a bioreactor. Biocatalysts are currently used to produce a wide range of products in the fields of food manufacture (such as bread, cheese, beer), fine chemicals (e.g., amino acids, vitamins), and pharmaceuticals (e.g., derivatives of antibiotics). They not only provide access to innovative products and processes, but also meet criteria of sustainability. In organic synthesis, recombinant technologies and biocatalysts have greatly widened the scope of application. Examples of current applications and processes are given. Recent developments and trends are presented as a survey, covering new methods for accessing biodiversity with new enzymes, directed evolution for improving enzymes, designed cells, and integrated downstream processing. [source] Crystallization for the Downstream Processing of ProteinsENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2005S. Schmidt Abstract Protein crystallization offers great potential in downstream processing of pharmaceutical protein active ingredients. The advantages, which are well known and widely utilized in low-molecular weight crystallization, can also be expected to be found to some extent in protein crystallization. However, there is still a marked need for improvement in two main areas of protein processing, namely, in crystallization from impure solutions and scale-up. [source] Purification of Aspergillus carbonarius polygalacturonase using polymeric membranesJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 7 2008E. Nakkeeran Abstract BACKGROUND: Microfiltration (MF: 70,450 nm) and ultrafiltration (UF: 10,500 kDa) membranes were used to eliminate carbohydrates and other non-protein impurities from Aspergillus carbonarius culture broth containing polygalacturonase enzyme (EC 3.2.1.15) that would otherwise interfere with the purification processes and lead to enzyme loss. Further, diafiltration was attempted to improve the elimination of impurities as well as recovery of enzymes. RESULTS: MF resulted in removal of 2,25% carbohydrates with an enzyme recovery of 69,82% from the crude culture broth owing to the secondary layer formation. UF with 10 kDa membrane eliminated most of the carbohydrates (96%), phosphate salts and total acids with a recovery of 96% polygalacturonase and resulted in greater productivity. Using the above procedure, the enzyme was concentrated nearly 10-fold while the purity improved from 4.6 to 49.4 U mg,1 of dry matter. CONCLUSIONS: The results of this study focused on the elimination of carbohydrates and other non-protein impurities showed that UF could be used efficiently as a primary purification step during downstream processing of microbial culture broths containing enzymes. The present approach will ensure complete elimination of non-protein impurities thereby reducing the losses and difficulties in the subsequent purification steps. Copyright © 2008 Society of Chemical Industry [source] The use of colloidal gas aphrons as novel downstream processing for the recovery of astaxanthin from cells of Phaffia rhodozymaJOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 2 2008Maria Dermiki Abstract BACKGROUND: There is an increasing interest in obtaining natural products with bioactive properties, using fermentation technology. However, the downstream processing consisting of multiple steps can be complicated, leading to increase in the final cost of the product. Therefore there is a need for integrated, cost-effective and scalable separation processes. RESULTS: The present study investigates the use of colloidal gas aphrons (CGA), which are surfactant-stabilized microbubbles, as a novel method for downstream processing. More particularly, their application for the recovery of astaxanthin from the cells of Phaffia rhodozyma is explored. Research carried out with standard solutions of astaxanthin and CGA generated from the cationic surfactant hexadecyl trimethyl ammonium bromide (CTAB) showed that up to 90% recovery can be achieved under optimum conditions, i.e., pH 11 with NaOH 0.2 mol L,1. In the case of the cells' suspension from the fermentation broth, three different approaches were investigated: (a) the conventional integrated approach where CGA were applied directly; (b) CGA were applied to the clarified suspension of cells; and finally (c) the in situ approach, where CGA are generated within the clarified suspension of cells. Interestingly, in the case of the whole suspension (approach a) highest recoveries (78%) were achieved under the same conditions found to be optimal for the standard solutions. In addition, up to 97% recovery of total carotenoids could be achieved from the clarified suspension after pretreatment with NaOH. This pretreatment led to maximum cell disruption as well as optimum conditioning for subsequent CGA separation. CONCLUSIONS: These results demonstrate the potential of CGA for the recovery of bioactive components from complex feedstock. Copyright © 2008 Society of Chemical Industry [source] OPTIMIZATION OF PERMEABILIZATION PROCESS FOR LACTOSE HYDROLYSIS IN WHEY USING RESPONSE SURFACE METHODOLOGYJOURNAL OF FOOD PROCESS ENGINEERING, Issue 3 2009GURPREET KAUR ABSTRACT To overcome the permeability barrier and prepare whole cell biocatalysts with high activities, permeabilization of Kluyveromyces marxianus var. lactis NCIM 3566 in relation to, -galactosidase activity was optimized using cetyltrimethylammonium bromide (CTAB) as permeabilizing agent. Permeabilized whole cells can be advantageous over pure enzyme preparations in terms of cost-effectiveness and increased stability maintained by the intracellular environment. Response surface methodology (RSM) was applied to optimize concentration of CTAB, temperature and the treatment time for maximum permeabilization of yeast cells. The optimum operating conditions for permeabilization process to achieve maximum enzyme activity obtained by RSM were 0.06% (w/v) CTAB concentration, 28C temperature and process duration of 14 min. At these conditions of process variables, the maximum value of enzyme activity was found to be 1,334 IU/g. The permeabilized yeast cells were highly effective and resulted in 90.5% lactose hydrolysis in whey. PRACTICAL APPLICATION , -Galactosidase is one of the most promising enzymes, which has several applications in the food, fermentation and dairy industry. However, the industrial applications of , -galactosidase have been hampered by the costs involved in downstream processing. The present investigation was focused on developing the low-cost technology for lactose hydrolysis based on permeabilization process. Disposal of lactose in whey and whey permeates is one of the most significant problems with regard to economics and environmental impact faced by the dairy industries. Keeping this in view, lactose hydrolysis in whey has been successfully performed using permeabilized Kluyveromyces marxianus cells. Hydrolysis of lactose using , -galactosidase converts whey into a potentially very useful food ingredient, which has immense applications in food industries. Its use has increased significantly in recent years, mainly in the dairy products and in digestive preparations. Lactose hydrolysis causes several potential changes in the manufacture and marketing of dairy products, including increased solubility, sweetness and broader fermentation possibilities. [source] A fast and inexpensive DNA extraction/purification protocol for brown macroalgaeMOLECULAR ECOLOGY RESOURCES, Issue 2 2007GALICE HOARAU Abstract Here we describe a rapid method for extracting DNA from dried brown algae material using a microtitre plate system in conjunction with a milling instrument. The method allows the preparation of nuclear and organelle DNA of quality suitable for polymerase chain reaction amplification. It combines high throughput with low cost per sample: DNA from 192 samples can be extracted in c. 3 h for < ,0.40 per sample, nearly tenfold cheaper than commercially available kits. Furthermore, by using microtitre plates, efficient storage and downstream processing is facilitated. [source] Efficient phase separation and product recovery in organic-aqueous bioprocessing using supercritical carbon dioxideBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010Christoph Brandenbusch Abstract Biphasic hydrocarbon functionalizations catalyzed by recombinant microorganisms have been shown to be one of the most promising approaches for replacing common chemical synthesis routes on an industrial scale. However, the formation of stable emulsions complicates downstream processing, especially phase separation. This fact has turned out to be a major hurdle for industrial implementation. To overcome this limitation, we used supercritical carbon dioxide (scCO2) for both phase separation and product purification. The stable emulsion, originating from a stereospecific epoxidation of styrene to (S)-styrene oxide, a reaction catalyzed by recombinant Escherichia coli, could be destabilized efficiently and irreversibly, enabling complete phase separation within minutes. By further use of scCO2 as extraction agent, the product (S)-styrene oxide could be obtained with a purity of 81% (w/w) in one single extraction step. By combining phase separation and product purification using scCO2, the number of necessary workup steps can be reduced to one. This efficient and easy to use technique is generally applicable for the workup of biphasic biocatalytic hydrocarbon functionalizations and enables a cost effective downstream processing even on a large scale. Biotechnol. Bioeng. 2010;107:642,651. © 2010 Wiley Periodicals, Inc. [source] Biomanufacturing process analytical technology (PAT) application for downstream processing: Using dissolved oxygen as an indicator of product quality for a protein refolding reactionBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Shelly A. Pizarro Abstract Process analytical technology (PAT) is an initiative from the US FDA combining analytical and statistical tools to improve manufacturing operations and ensure regulatory compliance. This work describes the use of a continuous monitoring system for a protein refolding reaction to provide consistency in product quality and process performance across batches. A small-scale bioreactor (3,L) is used to understand the impact of aeration for refolding recombinant human vascular endothelial growth factor (rhVEGF) in a reducing environment. A reverse-phase HPLC assay is used to assess product quality. The goal in understanding the oxygen needs of the reaction and its impact to quality, is to make a product that is efficiently refolded to its native and active form with minimum oxidative degradation from batch to batch. Because this refolding process is heavily dependent on oxygen, the % dissolved oxygen (DO) profile is explored as a PAT tool to regulate process performance at commercial manufacturing scale. A dynamic gassing out approach using constant mass transfer (kLa) is used for scale-up of the aeration parameters to manufacturing scale tanks (2,000,L, 15,000,L). The resulting DO profiles of the refolding reaction show similar trends across scales and these are analyzed using rpHPLC. The desired product quality attributes are then achieved through alternating air and nitrogen sparging triggered by changes in the monitored DO profile. This approach mitigates the impact of differences in equipment or feedstock components between runs, and is directly inline with the key goal of PAT to "actively manage process variability using a knowledge-based approach." Biotechnol. Bioeng. 2009; 104: 340,351 © 2009 Wiley Periodicals, Inc. [source] Microbial bio-production of a recombinant stimuli-responsive biosurfactantBIOTECHNOLOGY & BIOENGINEERING, Issue 1 2009W. Kaar Abstract Biosurfactants have been the subject of recent interest as sustainable alternatives to petroleum-derived compounds in areas ranging from soil remediation to personal and health care. The production of naturally occurring biosurfactants depends on the presence of complex feed sources during microbial growth and requires multicomponent enzymes for synthesis within the cells. Conversely, designed peptide surfactants can be produced recombinantly in microbial systems, enabling the generation of improved variants by simple genetic manipulation. However, inefficient downstream processing is still an obstacle for the biological production of small peptides. We present the production of the peptide biosurfactant GAM1 in recombinant E. coli. Expression was performed in fusion to maltose binding protein using chemically defined minimal medium, followed by a single-step affinity capture and enzymatic cleavage using tobacco etch virus protease. Different approaches to the isolation of peptide after cleavage were investigated, with special emphasis on rapid and simple procedures. Solvent-, acid-, and heat-mediated precipitation of impurities were successfully applied as alternatives to post-cleavage chromatographic peptide purification, and gave peptide purities exceeding 90%. Acid precipitation was the method of choice, due to its simplicity and the high purification factor and recovery rate achieved here. The functionality of the bio-produced peptide was tested to ensure that the resulting peptide biosurfactant was both surface active and able to be triggered to switch between foam-stabilizing and foam-destabilizing states. Biotechnol. Bioeng. 2009;102: 176,187. © 2008 Wiley Periodicals, Inc. [source] Micro biochemical engineering to accelerate the design of industrial-scale downstream processes for biopharmaceutical proteinsBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2008N.J. Titchener-Hooker Abstract The article examines how a small set of easily implemented micro biochemical engineering procedures combined with regime analysis and bioprocess models can be used to predict industrial scale performance of biopharmaceutical protein downstream processing. This approach has been worked on in many of our studies of individual operations over the last 10 years and allows preliminary evaluation to be conducted much earlier in the development pathway because of lower costs. It then permits the later large scale trials to be more highly focused. This means that the risk of delays during bioprocess development and of product launch are reduced. Here we draw the outcomes of this research together and illustrate its use in a set of typical operations; cell rupture, centrifugation, filtration, precipitation, expanded bed adsorption, chromatography and for common sources, E. coli, two yeasts and mammalian cells (GS-NSO). The general approach to establishing this method for other operations is summarized and new developments outlined. The technique is placed against the background of the scale-down methods that preceded it and complementary ones that are being examined in parallel. The article concludes with a discussion of the advantages and limitations of the micro biochemical engineering approach versus other methods. Biotechnol. Bioeng. 2008;100: 473,487. © 2008 Wiley Periodicals, Inc. [source] Viral clearance using disposable systems in monoclonal antibody commercial downstream processingBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2008Joe X. Zhou Abstract Once highly selective protein A affinity is chosen for robust mAb downstream processing, the major role of polishing steps is to remove product related impurities, trace amounts of host cell proteins, DNA/RNA, and potential viral contaminants. Disposable systems can act as powerful options either to replace or in addition to polishing column chromatography to ensure product purity and excellent viral clearance power for patients' safety. In this presentation, the implementation of three disposable systems such as depth filtration, membrane chromatography, and nanometer filtration technology in a commercial process are introduced. The data set of viral clearance with these systems is presented. Application advantages and disadvantages including cost analysis are further discussed. Biotechnol. Bioeng. 2008;100: 488,496. © 2008 Wiley Periodicals, Inc. [source] A method for lipase co-precipitation in a biodegradable protein matrixBIOTECHNOLOGY & BIOENGINEERING, Issue 6 2007M. Golubovic Abstract This article presents a novel method for immobilization of active ingredients. The method is based on CO2 aided active ingredient co-precipitation with glycinin, a biodegradable protein matrix from edible soybean protein. Glycinin precipitates abundantly under isoelectric conditions and serves as the matrix within which the active substance is trapped during the precipitation process. The enzyme lipase from Candida rugosa was successfully co-precipitated into the protein pellet to prove the principle. It was shown that the lipase within the co-precipitate retained lipase and esterase activity under different pH conditions. In some cases the activity was even higher than the activity of crude lipase, possibly due to the protective role of the matrix protein. Due to the retained lipase activity and food-grade quality of the binary precipitate, it has potential of being used in the food or pharmaceutical industry. Additional quality of the binary precipitate is the potentially significantly reduced downstream processing due to the fact that no organic solvents or precipitants were used in the precipitation process. Biotechnol. Bioeng. 2007;98: 1209,1218. © 2007 Wiley Periodicals, Inc. [source] The influence of homogenisation conditions on biomass-adsorbent interactions during ion-exchange expanded bed adsorptionBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2006Jürgen J. Hubbuch Expanded bed adsorption (EBA) is an integrative step in downstream processing allowing the direct capture of target proteins from cell-containing feedstocks. Extensive co-adsorption of biomass, however, may hamper the application of this technique. The latter is especially observed at anion exchange processes as cells or cell debris are negatively charged under common anion exchange conditions. The restrictions observed under these conditions are, however, directly related to processing steps prior to fluidised bed application. In this study, it could be shown that the effective surface charge of cell debris obtained during homogenisation is closely related to the debris size and thus to the homogenisation method and conditions. The amount and thus effect of cells binding to the adsorbent could be significantly decreased when optimising the homogenisation step not only towards optimal product release but towards a reduction of debris size and charge. The lower size and charge of the debris results not only in a reduced retention probability but also, in a lower collision probability between debris and adsorbent. The applicability was shown in an example where the homogenisation conditions of E. coli were optimised towards EBA applications. In a previous report (Reichert et al., 2001) studying the suitability of EBA for the capture of formate dehydrogenate from E. coli homogenate the pseudo affinity resin Streamline Red was identified as the only suitable adsorbent. The new approach, however, led to a system where anion exchange as capture step became possible, however, to the cost of binding capacity. © 2006 Wiley Periodicals, Inc. [source] Affinity-enhanced protein partitioning in decyl ,- D -glucopyranoside two-phase aqueous micellar systemsBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2005Henry Lam Abstract Liquid,liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products. In the case of two-phase aqueous micellar systems, careful choices of the phase-forming surfactants or surfactant mixtures allow these systems to separate biomolecules based on size, hydrophobicity, charge, or specific affinity. In this article, we investigate the affinity-enhanced partitioning of a model affinity-tagged protein,green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP),in a two-phase aqueous micellar system generated from the nonionic surfactant n -decyl ,- D -glucopyranoside (C10G1), which acts simultaneously as the phase-former and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, despite the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a sixfold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp), as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein. To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain. © 2005 Wiley Periodicals, Inc. [source] Cover Picture: Biotechnology Journal 6/2010BIOTECHNOLOGY JOURNAL, Issue 6 2010Article first published online: 1 JUN 2010 Cover illustration: Biotech Methods and Advances. This issue of Biotechnology Journal covers methods for systems metabolic engineering as well as downstream processing. The cover image shows an artist's impression of a magnetic fusion protein consisting of antibody like particles (pale blue) and ferritin subunits (pale green) encapsulating an iron core (red). These "Magnetizable antibody-like proteins" are described by Dehal et al. pp. 596,605 (http://dx.doi.org/10.1002/biot.200900273). Art by Mark Bradshaw, Pixelweave 3D. [source] Industrial biotechnology: Tools and applicationsBIOTECHNOLOGY JOURNAL, Issue 12 2009Weng Lin Tang Abstract Industrial biotechnology involves the use of enzymes and microorganisms to produce value-added chemicals from renewable sources. Because of its association with reduced energy consumption, greenhouse gas emissions, and waste generation, industrial biotechnology is a rapidly growing field. Here we highlight a variety of important tools for industrial biotechnology, including protein engineering, metabolic engineering, synthetic biology, systems biology, and downstream processing. In addition, we show how these tools have been successfully applied in several case studies, including the production of 1, 3-propanediol, lactic acid, and biofuels. It is expected that industrial biotechnology will be increasingly adopted by chemical, pharmaceutical, food, and agricultural industries. [source] Disaggregation of high-molecular weight species during downstream processing to recover functional monomerBIOTECHNOLOGY PROGRESS, Issue 3 2010Xuankuo Xu Abstract The use of chaotropic agents to recover functional monomeric material was investigated for the downstream purification of an Fc-fusion protein containing high levels of high-molecular weight (HMW) species. In batch studies, chaotropic agents irreversibly disaggregated a majority of the aggregated protein. An integrated processing mode, termed as on-column disaggregation, was developed in which the protein was captured on Protein A chromatography and then a chaotropic agent was used to simultaneously elute the bound protein and disaggregate the HMW species. On-column disaggregation process resulted in protein recoveries of >95% and aggregation reduction of ,50%. Analytical results are presented showing that the recovered monomeric material was comparable to the reference protein in biochemical, biophysical, and pharmacokinetic properties. The kinetic and molecular mechanisms governing protein aggregation and disaggregation will also be elucidated. For the Fc-fusion protein studied here, incorporation of the disaggregation strategy in both batch and on-column modes led to an increase of >10% in overall downstream yield. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Mcl-1 overexpression leads to higher viabilities and increased production of humanized monoclonal antibody in Chinese hamster ovary cellsBIOTECHNOLOGY PROGRESS, Issue 4 2009Brian S. Majors Abstract Bioreactor stresses, including nutrient deprivation, shear stress, and byproduct accumulation can cause apoptosis, leading to lower recombinant protein yields and increased costs in downstream processing. Although cell engineering strategies utilizing the overexpression of antiapoptotic Bcl-2 family proteins such as Bcl-2 and Bcl-xL potently inhibit apoptosis, no studies have examined the use of the Bcl-2 family protein, Mcl-1, in commercial mammalian cell culture processes. Here, we overexpress both the wild type Mcl-1 protein and a Mcl-1 mutant protein that is not degraded by the proteasome in a serum-free Chinese hamster ovary (CHO) cell line producing a therapeutic antibody. The expression of Mcl-1 led to increased viabilities in fed-batch culture, with cell lines expressing the Mcl-1 mutant maintaining ,90% viability after 14 days when compared with 65% for control cells. In addition to enhanced culture viability, Mcl-1-expressing cell lines were isolated that consistently showed increases in antibody production of 20,35% when compared with control cultures. The quality of the antibody product was not affected in the Mcl-1-expressing cell lines, and Mcl-1-expressing cells exhibited 3-fold lower caspase-3 activation when compared with the control cell lines. Altogether, the expression of Mcl-1 represents a promising alternative cell engineering strategy to delay apoptosis and increase recombinant protein production in CHO cells. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Emergence of Ideal Membrane Cascades for Downstream ProcessingBIOTECHNOLOGY PROGRESS, Issue 3 2008Edwin N. Lightfoot An algorithm is developed for describing ideal membrane cascades for fractionation of binary and pseudo-binary mixtures. It is shown that solvent management plays a key role in determining both purification and yield. Development of efficient diafilters is needed if membrane cascades are to achieve their full potential in competing with both chromatography and simulated moving bed operations in downstream processing of proteins. Such a replacement will also be important for fractionation of higher titers and larger substrates, such as plasmids, viruses, and even whole cells. [source] Applying Near-Infrared Spectroscopy in Downstream Processing: One Calibration for Multiple Clarification Processes of Fermentation MediaBIOTECHNOLOGY PROGRESS, Issue 2 2008Licínia O. Rodrigues The use of near-infrared spectroscopy (NIRS) is demonstrated in the first downstream processing (DSP) steps of an active pharmaceutical ingredient (API) manufacturing process. The first method developed was designed to assess the API content in the filtrate stream (aqueous) of a rotary drum vacuum filter. The PLS method, built after spectral preprocessing and variable selection, had an accuracy of 0.01% (w/w) for an API operational range between 0.20 and 0.45% (w/w). The robustness and extrapolation ability of the calibration was proved when samples from ultrafiltration and nanofiltration processes, ranging from 0 to 2% (w/w), were linearly predicted ( R2=0.99). The development of a robust calibration model is generally a very time-consuming task, and once established it is imperative that it can be useful for a long period of time. This work demonstrates that NIR procedures, when carefully developed, can be used in different process conditions and even in different process steps of similar unit operations. [source] Process Technology for Production and Recovery of Heterologous Proteins with Pichia pastorisBIOTECHNOLOGY PROGRESS, Issue 6 2006Mehmedalija Jahic Developments in process techniques for production and recovery of heterologous proteins with Pichia pastoris are presented. Limitations for the standard techniques are described, and alternative techniques that solve the limitations problems are reviewed together with the methods that resulted in higher productivity of the P. pastoris processes. The main limitations are proteolysis of the secreted products and cell death in the high cell density bioreactor cultures. As a consequence, both low productivity and lower quality of the feedstock for downstream processing are achieved in processes hampered with these problems. Methods for exploring proteolysis and cell death are also presented. Solving the problems makes the conditions for downstream processing superior for the P. pastoris expression systems compared to other systems, which either need complex media or rely on intracellular production. These improved conditions allow for interfacing of cultivation with downstream processing in an integrated fashion. [source] Optimization of Isopropanol and Ammonium Sulfate Precipitation Steps in the Purification of Plasmid DNABIOTECHNOLOGY PROGRESS, Issue 4 2006S. S. Freitas Large-scale processes used to manufacture grams of plasmid DNA (pDNA) should be cGMP compliant, economically feasible, and environmentally friendly. Alcohol and salt precipitation techniques are frequently used in plasmid DNA (pDNA) downstream processing, as concentration and prepurification steps, respectively. This work describes a study of a standard 2-propanol (IsopOH; 0.7 v/v) and ammonium sulfate (AS; 2.5 M) precipitation. When inserted in a full process, this tandem precipitation scheme represents a high economic and environmental impact due to the large amounts of the two precipitant agents and their environmental relevance. Thus, major goals of the study were the minimization of precipitants and the selection of the best operating conditions for high pDNA recovery and purity. The pDNA concentration in the starting Escherichia coli alkaline lysate strongly affected the efficiency of IsopOH precipitation as a concentration step. The results showed that although an IsopOH concentration of at least 0.6 (v/v) was required to maximize recovery when using lysates with less than 80 ,g pDNA/mL, concentrations as low as 0.4 v/v could be used with more concentrated lysates (170 ,g pDNA/mL). Following resuspension of pDNA pellets generated by 0.6 v/v IsopOH, precipitation at 4 °C with 2.4 M AS consistently resulted in recoveries higher than 80% and in removal of more than 90% of the impurities (essentially RNA). An experimental design further indicated that AS concentrations could be reduced down to 2.0 M, resulting in an acceptable purity (21,23%) without compromising recovery (84,86%). Plasmid recovery and purity after the sequential IsopOH/AS precipitation could be further improved by increasing the concentration factor (CF) upon IsopOH precipitation from 2 up to 25. Under these conditions, IsopOH and AS concentrations of 0.60 v/v and 1.6 M resulted in high recovery (,100%) and purity (32%). In conclusion, it is possible to reduce substantially the mass of precipitation agents used without affecting recovery, if a small concession is made regarding purity. This directly translates into an improvement of the process economics and in a reduction of the environmental impact of the process. [source] Improved Methods for Prepurification and Detection of Staphylococcal Enterotoxin B from Cell-Free Culture FiltrateBIOTECHNOLOGY PROGRESS, Issue 4 2005Maria Dainiak An improved ELISA method for the detection of Staphylococcal Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery. [source] Antibodies and Genetically Engineered Related Molecules: Production and PurificationBIOTECHNOLOGY PROGRESS, Issue 3 2004A. Cecília A. Roque Antibodies and antibody derivatives constitute 20 % of biopharmaceutical products currently in development, and despite early failures of murine products, chimeric and humanized monoclonal antibodies are now viable therapeutics. A number of genetically engineered antibody constructions have emerged, including molecular hybrids or chimeras that can deliver a powerful toxin to a target such as a tumor cell. However, the general use in clinical practice of antibody therapeutics is dependent not only on the availability of products with required efficacy but also on the costs of therapy. As a rule, a significant percentage (50,80%) of the total manufacturing cost of a therapeutic antibody is incurred during downstream processing. The critical challenges posed by the production of novel antibody therapeutics include improving process economics and efficiency, to reduce costs, and fulfilling increasingly demanding quality criteria for Food and Drug Administration (FDA) approval. It is anticipated that novel affinity-based separations will emerge from the development of synthetic ligands tailored to specific biotechnological needs. These synthetic affinity ligands include peptides obtained by synthesis and screening of peptide combinatorial libraries and artificial non-peptidic ligands generated by a de novo process design and synthesis. The exceptional stability, improved selectivity, and low cost of these ligands can lead to more efficient, less expensive, and safer procedures for antibody purification at manufacturing scales. This review aims to highlight the current trends in the design and construction of genetically engineered antibodies and related molecules, the recombinant systems used for their production, and the development of novel affinity-based strategies for antibody recovery and purification. [source] | |||||||||||||