Showing papers in "Biotechnology Progress in 2006"

Synthesis of gold nanotriangles and silver nanoparticles using Aloe vera plant extract.

Abstract: Biogenic gold nanotriangles and spherical silver nanoparticles were synthesized by a simple procedure using Aloe vera leaf extract as the reducing agent. This procedure offers control over the size of the gold nanotriangle and thereby a handle to tune their optical properties, particularly the position of the longitudinal surface plasmon resonance. The kinetics of gold nanotriangle formation was followed by UV-vis-NIR absorption spectroscopy and transmission electron microscopy (TEM). The effect of reducing agent concentration in the reaction mixture on the yield and size of the gold nanotriangles was studied using transmission electron microscopy. Monitoring the formation of gold nanotriangles as a function of time using TEM reveals that multiply twinned particles (MTPs) play an important role in the formation of gold nanotriangles. It is observed that the slow rate of the reaction along with the shape directing effect of the constituents of the extract are responsible for the formation of single crystalline gold nanotriangles. Reduction of silver ions by Aloe vera extract however, led to the formation of spherical silver nanoparticles of 15.2 nm +/- 4.2 nm size.

Microalgal Reactors: A Review of Enclosed System Designs and Performances

Abstract: One major challenge to industrial microalgal culturing is to devise and develop technical apparata, cultivation procedures and algal strains susceptible of undergoing substantial increases in efficiency of use of solar energy and carbon dioxide. Despite several research efforts developed to date, there is no such thing as "the best reactor system"- defined, in an absolute fashion, as the one able to achieve maximum productivity with minimum operation costs, irrespective of the biological and chemical system at stake. In fact, choice of the most suitable system is situation-dependent, as both the species of alga available and the final purpose intended will play a role. The need of accurate control impairs use of open-system configurations, so current investigation has focused mostly on closed systems. In this review, several types of closed bioreactors described in the technical literature as able to support production of microalgae are comprehensively presented and duly discussed, using transport phenomenon and process engineering methodological approaches. The text is subdivided into subsections on: reactor design, which includes tubular reactors, flat plate reactors and fermenter-type reactors; and processing parameters, which include gaseous transfer, medium mixing and light requirements.

From 3D to 2D: a review of the molecular imprinting of proteins.

Abstract: Molecular imprinting is a generic technology that allows for the introduction of sites of specific molecular affinity into otherwise homogeneous polymeric matrices. Commonly this technique has been shown to be effective when targeting small molecules of molecular weight <1500, while extending the technique to larger molecules such as proteins has proven difficult. A number of key inherent problems in protein imprinting have been identified, including permanent entrapment, poor mass transfer, denaturation, and heterogeneity in binding pocket affinity, which have been addressed using a variety of approaches. This review focuses on protein imprinting in its various forms, ranging from conventional bulk techniques to novel thin film and monolayer surface imprinting approaches.

Ethanol production from alkaline peroxide pretreated enzymatically saccharified wheat straw.

Abstract: Wheat straw used in this study contained 44.24 +/- 0.28% cellulose and 25.23 +/- 0.11% hemicellulose. Alkaline H(2)O(2) pretreatment and enzymatic saccharification were evaluated for conversion of wheat straw cellulose and hemicellulose to fermentable sugars. The maximum yield of monomeric sugars from wheat straw (8.6%, w/v) by alkaline peroxide pretreatment (2.15% H(2)O(2), v/v; pH 11.5; 35 degrees C; 24 h) and enzymatic saccharification (45 degrees C, pH 5.0, 120 h) by three commercial enzyme preparations (cellulase, beta-glucosidase, and xylanase) using 0.16 mL of each enzyme preparation per g of straw was 672 +/- 4 mg/g (96.7% yield). During the pretreatment, no measurable quantities of furfural and hydroxymethyl furfural were produced. The concentration of ethanol (per L) from alkaline peroxide pretreated enzyme saccharified wheat straw (66.0 g) hydrolyzate by recombinant Escherichia coli strain FBR5 at pH 6.5 and 37 degrees C in 48 h was 18.9 +/- 0.9 g with a yield of 0.46 g per g of available sugars (0.29 g/g straw). The ethanol concentration (per L) was 15.1 +/- 0.1 g with a yield of 0.23 g/g of straw in the case of simultaneous saccharification and fermentation by the E. coli strain at pH 6.0 and 37 degrees C in 48 h.

Role of Caco‐2 Cell Monolayers in Prediction of Intestinal Drug Absorption

Abstract: The usefulness of Caco-2 cell monolayers in determining the intestinal drug absorption of potential drug candidates as such and from delivery systems, elucidating the underlying mechanisms thereof, presystemic metabolism, cellular uptake and cytotoxicological assessment has been exemplified in this review. The role of Caco-2 cell monolayers in studying the effectiveness, involved mechanism and toxicity of various excipients for drug absorption promotion has also been discussed.

Basic Concepts in Q Membrane Chromatography for Large‐Scale Antibody Production

Abstract: The large-scale production of recombinant human monoclonal antibodies demands economical purification processes with high throughputs. In this article we briefly describe a common antibody process and evaluate the Q membrane adsorber for process-scale antibody production as an alternative to a Q-packed-bed column in a flow-through mode. The scientific concepts underlining Q membrane technology and its application are reviewed. The disadvantages and advantages of using Q membrane chromatography as a purification unit in large-scale production are discussed, including problems initially seen with the Q membrane scale-down model but solved with the invention of a new scale-down model. The new Q-membrane unit operation has a process capacity greater than 3,000 g/m(2) or 10.7 kg/L with a LRV over 5 for four model viruses. In this Review, a cost analysis illustrates that Q membrane chromatography is a viable alternative to Q column chromatography as a polishing step in a flow-through mode for process-scale antibody production.

Exploitation of the adsorptive properties of depth filters for host cell protein removal during monoclonal antibody purification.

Abstract: Depth filtration has been widely used during process scale clarification of cell culture supernatants for the removal of cells and cell debris. However, in addition to their filtration capabilities, depth filters also possess the ability to adsorb soluble species. This aspect of depth filtration has largely not been exploited in process scale separations and is usually ignored during cell culture harvest development. Here, we report on the ability of depth filters to adsorptively remove host cell protein contaminants from a recombinant monoclonal antibody process stream and characterize some of the underlying interactions behind the binding phenomenon. Following centrifugation, filtration through a depth filter prior to Protein A chromatographic capture was shown to significantly reduce the level of turbidity observed in the Protein A column eluate of the monoclonal antibody. The Protein A eluate turbidity was shown to be linked to host cell protein contaminant levels in the Protein A column load and not to the DNA content. Analogous to flowthrough chromatography in which residence time/bed height and column loading are key parameters, both the number of passes through the depth filter and the amount of centrifuge centrate loaded on the filter were seen to be important operational parameters governing the adsorptive removal of host cell protein contaminants. Adsorption of proteins to the depth filter was shown to be due to a combination of electrostatic and hydrophobic adsorptive interactions. These results demonstrate the ability to employ depth filtration as an integrative unit operation combining filtration for particulate removal with adsorptive binding for contaminant removal.

One-step, painting-like coating procedures to make surfaces highly and permanently bactericidal.

Abstract: Previously we found that covalent attachment of long-chained, moderately hydrophobic polycations to surfaces of solid objects renders the latter permanently bactericidal. Herein we replaced such surface-specific, multistep immobilization techniques with a single-step, general procedure akin to common painting. Glass or polyethylene slides were briefly dipped into organic solutions of certain optimally hydrophobic N-alkyl-PEI (where PEI stands for branched 750-kDa polyethylenimine) polycations, followed by solvent evaporation. The resultant polycation-coated slides were able to kill on contact all of the encountered bacterial cells, whether the Gram-positive human pathogen Staphylococcus aureus or its Gram-negative brethren Escherichia coli. This biocidal effect was found not to be caused by N-alkyl-PEI molecules leached from the surface. Further examination of the mechanism of this bactericidal action suggested that the surface-deposited N-alkyl-PEI kills bacteria by rupturing their cellular membranes. This conclusion was further supported by studies in which the molecular weight of PEI and the hydrophobicity of the alkyl moiety were varied.

Butanol production from corn fiber xylan using Clostridium acetobutylicum.

Abstract: Acetone, butanol, and ethanol (ABE) were produced from corn fiber arabinoxylan (CFAX) and CFAX sugars (glucose, xylose, galactose, and arabinose) using Clostridium acetobutylicum P260. In mixed sugar (glucose, xylose, galactose, and arabinose) fermentation, the culture preferred glucose and arabinose over galactose and xylose. Under the experimental conditions, CFAX (60 g/L) was not fermented until either 5 g/L xylose or glucose plus xylanase enzyme were added to support initial growth and fermentation. In this system, C. acetobutylicum produced 9.60 g/L ABE from CFAX and xylose. This experiment resulted in a yield and productivity of 0.41 and 0.20 g/L x h, respectively. In the integrated hydrolysis, fermentation, and recovery process, 60 g/L CFAX and 5 g/L xylose produced 24.67 g/L ABE and resulted in a higher yield (0.44) and a higher productivity (0.47 g/L x h). CFAX was hydrolyzed by xylan-hydrolyzing enzymes, and ABE were recovered by gas stripping. This investigation demonstrated that integration of hydrolysis of CFAX, fermentation to ABE, and recovery of ABE in a single system is an economically attractive process. It is suggested that the culture be further developed to hydrolyze CFAX and utilize all xylan sugars simultaneously. This would further increase productivity of the reactor.

Efficiency of new fungal cellulase systems in boosting enzymatic degradation of barley straw lignocellulose.

Abstract: This study examined the cellulytic effects on steam-pretreated barley straw of cellulose-degrading enzyme systems from the five thermophilic fungi Chaetomium thermophilum, Thielavia terrestris, Thermoascus aurantiacus, Corynascus thermophilus, and Myceliophthora thermophila and from the mesophile Penicillum funiculosum. The catalytic glucose release was compared after treatments with each of the crude enzyme systems when added to a benchmark blend of a commercial cellulase product, Celluclast, derived from Trichoderma reesei and a beta-glucosidase, Novozym 188, from Aspergillus niger. The enzymatic treatments were evaluated in an experimental design template comprising a span of pH (3.5-6.5) and temperature (35-65 degrees C) reaction combinations. The addition to Celluclast + Novozym 188 of low dosages of the crude enzyme systems, corresponding to 10 wt % of the total enzyme protein load, increased the catalytic glucose yields significantly as compared to those obtained with the benchmark Celluclast + Novozyme 188 blend. A comparison of glucose yields obtained on steam-pretreated barley straw and microcrystalline cellulose, Avicel, indicated that the yield improvements were mainly due to the presence of highly active endoglucanase activity/activities in the experimental enzyme preparations. The data demonstrated the feasibility of boosting the widely studied T. reeseicellulase enzyme system with additional enzymatic activity to achieve faster lignocellulose degradation. We conclude that this supplementation strategy appears feasible as a first step in identifying truly promising fungal enzyme sources for fast development of improved, commercially viable, enzyme preparations for lignocellulose degradation.

Development of a Temperature‐Sensitive Composite Hydrogel for Drug Delivery Applications

Abstract: To develop materials with improved controllability and specificity, we have investigated composite hydrogels with temperature-sensitive properties using photo cross-linking. Specifically, our novel composite materials are composed of nanoparticles made of poly(N-isopropylacrylamide) (PNIPAAm), temperature-sensitive hydrogels, and a photo cross-linker, poly(ethylene glycol) diacrylate (PEGDA). PNIPAAm particles were synthesized by emulsion polymerization and by varying concentration of four main factors: monomers (N-isopropylacrylamide), cross-linkers (N,N'-methylenebisacrylamide), surfactants (sodium dodecyl sulfate, SDS), and initiators (potassium persulfate). We found that the surfactant, SDS, was the most important factor affecting the particle size using the factorial design analysis. Additionally, both nano- and micro-PNIPAAm particles had excellent loading efficiency (>80% of the incubated bovine serum albumin (BSA)), and their release kinetics expressed an initial burst effect followed by a sustained release over time. Furthermore, BSA-loaded PNIPAAm nanoparticles were used to form three-dimensional gel networks by means of a photocuring process using a photo cross-linker, PEGDA, and a photoinitiator, Irgacure-2959 (I-2959). Results from scanning electron microscopy and in vitro BSA release studies from these hydrogels demonstrated that PNIPAAm nanoparticles were embedded inside the PEG polymeric matrix and the composite material was able to release BSA in response to changes in temperature. These PNIPAAm nanoparticle hydrogel networks may have advantages in applications of controlled drug delivery systems because of their temperature sensitivity and their ability of in situ photopolymerization to localize at the specific region in the body.

Energy and emission benefits of alternative transportation liquid fuels derived from switchgrass: a fuel life cycle assessment.

Abstract: We conducted a mobility chains, or well-to-wheels (WTW), analysis to assess the energy and emission benefits of cellulosic biomass for the U.S. transportation sector in the years 2015-2030. We estimated the life-cycle energy consumption and emissions associated with biofuel production and use in light-duty vehicle (LDV) technologies by using the Greenhouse gases, Regulated Emissions, and Energy use in Transportation (GREET) model. Analysis of biofuel production was based on ASPEN Plus model simulation of an advanced fermentation process to produce fuel ethanol/protein, a thermochemical process to produce Fischer-Tropsch diesel (FTD) and dimethyl ether (DME), and a combined heat and power plant to co-produce steam and electricity. Our study revealed that cellulosic biofuels as E85 (mixture of 85% ethanol and 15% gasoline by volume), FTD, and DME offer substantial savings in petroleum (66-93%) and fossil energy (65-88%) consumption on a per-mile basis. Decreased fossil fuel use translates to 82-87% reductions in greenhouse gas emissions across all unblended cellulosic biofuels. In urban areas, our study shows net reductions for almost all criteria pollutants, with the exception of carbon monoxide (unchanged), for each of the biofuel production option examined. Conventional and hybrid electric vehicles, when fueled with E85, could reduce total sulfur oxide (SO x ) emissions to 39-43% of those generated by vehicles fueled with gasoline. By using bio-FTD and bio-DME in place of diesel, SO x emissions are reduced to 46-58% of those generated by diesel-fueled vehicles. Six different fuel production options were compared. This study strongly suggests that integrated heat and power co-generation by means of gas turbine combined cycle is a crucial factor in the energy savings and emission reductions.

Examination of mineralized nodule formation in living osteoblastic cultures using fluorescent dyes.

Abstract: Detecting the formation of mineralized nodules in osteogenic cell culture provides a means of assessing mature osteoblast cell function and the status of culture. In the present study, to continuously monitor the formation of mineralized nodules during the entire culture period, different concentrations of two fluorescent dyes (xylenol orange and calcein blue) were evaluated for their ability to specifically label calcified areas and their toxicity to cells in osteogenic cultures. Results showed that 20 microM xylenol orange and 30 microM calcein blue gave rise to distinct fluorescent staining for mineralized nodules, which were correlated exactly with von Kossa and alizarin red S staining at the same locations in cultures. In the assessment of toxicity, both dyes at the aforementioned concentrations did not alter cell viability or change the total DNA content in cultures. To demonstrate the advantage of using these fluorochromes to monitor mineralized nodules formation, consecutive fluorescent images of each staining were recorded at the same location of individual culture over the entire duration. The result indicates that both xylenol orange and calcein blue can provide contrasting fluorescent staining to continuously monitor mineralized nodules formation in living osteogenic cell cultures without deleterious effects.

Process Technology for Production and Recovery of Heterologous Proteins with Pichia pastoris

Abstract: 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.

Regulation of Recombinant Monoclonal Antibody Production in Chinese Hamster Ovary Cells: A Comparative Study of Gene Copy Number, mRNA Level, and Protein Expression

Abstract: We performed a comparative analysis on CHO cells with varying recombinant monoclonal antibody production rates (qAb) and investigated the regulation of MAb production. Two families of CHO cells each composed of one parental and two progeny cell lines, generated by stepwise increases in methotrexate (MTX) concentration, were studied. The MAb heavy chain (HC) and light (LC) gene copy numbers and mRNA levels were quantitated by Southern and northern blotting in midexponential growth phase. We observed 2- to 3-fold amplification in gene copy numbers in high producing cell lines when compared with parental cell lines. However, the mRNA levels in the high producers were 5- to 7-fold higher, correlating well with the 5- to 7-fold increase in qAb. These results were confirmed by real-time qPCR analysis. Our study reveals that the MTX-mediated MAb overexpression results from both an increase in the gene copy number and more efficient transcription of each gene copy. We also observed that the HC to LC gene copy ratio may affect the protein yield in MAb expression systems.

Ultra‐High Expression of a Thermally Responsive Recombinant Fusion Protein in E. coli

Abstract: Elastin-like polypeptides (ELPs) are recombinant peptide-based biopolymers that contain repetitive sequences enriched in glycine, valine, proline, and alanine. Because of the unusually large fraction of these amino acids in ELPs as compared to other cellular proteins, we hypothesized that intracellular pools of these amino acids can be selectively depleted and limit protein yields during expression. In this study, we examined how culture conditions and individual medium components affect protein yields by monitoring cell growth and protein expression kinetics of E. coli expressing an ELP tagged with a green fluorescent protein (GFP). By determining the underlying principles of superior fusion protein yields generated by the hyperexpression protocol, we further improved protein yields through the addition of glycerol and certain amino acids such as proline and alanine and found that amino acid concentrations and the type of basal medium used strongly influenced this beneficial effect. Surprisingly, amino acids other than those that are abundant in ELPs, for example, asparagine, aspartic acid, glutamine, and glutamic acid, also enhanced protein yields even in a nutrient-rich medium. Compared to commonly used Luria-Bertani medium, the protein yield was improved by 36-fold to the remarkable level of 1.6 g/L in shaker flask cultures with a modified medium and optimized culture conditions, which also led to a 8-fold reduction in the cost of the fusion protein. To our knowledge, this is the highest yield of an ELP-fusion protein purified from E. coli cultured in shaker flasks. This study also suggests a useful strategy to improve the yields of other ELP fusion proteins and repetitive polypeptides.

Surface Functionalized Electrospun Biodegradable Nanofibers for Immobilization of Bioactive Molecules

Abstract: A blend mixture of biodegradable poly(epsilon-caprolactone) (PCL) and poly(d,l-lactic-co-glycolic acid)-poly(ethylene glycol)-NH(2) (PLGA-b-PEG-NH(2)) block copolymer was electrospun to produce surface functionalized nanofibers. The resulting nanofibrous mesh with primary amine groups on the surface was applied for immobilization of biologically active molecules using lysozyme as a model enzyme. Lysozyme was immobilized via covalent conjugation by using a homobifunctional coupling agent. The nanofibrous mesh could immobilize a far greater amount of lysozyme on the surface with concomitantly increased activity, primarily due to its larger surface area, compared to that of the solvent casting film. It was also found that the enzyme immobilization process slightly altered thermal and pH-dependent catalytic activity profiles compared to those of native lysozyme. The results demonstrated the surface functionalized electrospun nanofibrous mesh could be used as a promising material for immobilizing a wide range of bioactive molecules.

Effect of overexpression of a soluble pyridine nucleotide transhydrogenase (UdhA) on the production of poly(3-hydroxybutyrate) in Escherichia coli.

Abstract: A soluble pyridine nucleotide transhydrogenase (UdhA) has been used to increase the productivity and yield of PHB in vivo. By inducing a high level of UdhA, which can transfer reducing equivalents between NAD and NADP, we have increased NADPH availability, resulting in high yield and productivity of PHB in Escherichia coli. Coexpression of the phb operon from Alcaligenes eutrophus H16 and the native udhA from E. coli from high copy plasmids resulted in an increase in PHB yield from 49 to 66% g of PHB per gram of total cell dry weight and an increase in final concentration from 3.52 to 6.42 g/L; the PHB concentration of the udhA carrying strain is almost twice that of the control strain expressing only the phb operon. The results of this study demonstrate the effectiveness of cofactor manipulation and its application as a tool in metabolic engineering.

A comparison of air and hydrogen peroxide oxygenated microbial fuel cell reactors.

Abstract: In this study, a two-compartment continuous flow microbial fuel cell (MFC) reactor was used to compare the efficiencies of cathode oxygenation by air and by hydrogen peroxide. The MFC reactor had neither a proton-selective membrane nor an electron transfer mediator. At startup, the cathodic compartment was continuously aerated and the anodic compartment was fed with a glucose solution. An increase of electrical power generation from 0.008 to 7.2 mW m(-2) of anode surface with a steady-state potential of 215-225 mV was observed within a period of 12 days. The performance of the air-oxygenated MFC reactor progressively declined over time because of biofilm proliferation in the cathodic compartment. Oxygenation of the cathodic compartment using 300 mL d(-1) of 0.3% hydrogen peroxide solution resulted in a power density of up to 22 mW m(-2) (68.2 mA m(-2)) of anode surface at a potential of 340-350 mV. The use of H2O2 for oxygenation was found to improve the long-term stability of the MFC reactor.

Cost-effective recovery and purification of polyhydroxyalkanoates by selective dissolution of cell mass.

Abstract: Highly efficient separation and purification of polyhydroxyalkanoates (PHAs) from PHA-containing cell mass is essential to production of the bioplastics from renewable resources in a cost-effective, environmentally friendly way. Based on selective dissolution of non-PHA cell mass (NPCM) by protons in aqueous solution and crystallization kinetics of PHA biopolymers, a simple process is developed and demonstrated to recover PHAs from cell mass to high purity (>97 wt %) with high yield (>95 wt %). The average molecular weight of biopolyesters is controlled, which follows an exponential function of process severity, a combined factor of processing conditions. Compared with conventional chemical treatment such as sequential surfactant and hypochlorite treatment, this new technology substantially reduces the chemical cost for PHA recovery and purification from PHA-containing cell mass.

Growth of Human Keratinocytes and Fibroblasts on Bacterial Cellulose Film

Abstract: Thin films of bacterial cellulose (BC) from a nata de coco culture system were developed, characterized, and investigated for the growth of human keratinocytes and fibroblasts. The average pore diameter and total surface area of the dried BC films estimated by BET were 224 A and 12.62 m(2)/g, respectively. With an film thickness of 0.12 mm, the average tensile strength and break strain of the dried films were 5.21 MPa and 3.75%, whereas those of the wet films were 1.56 MPa and 8.00%, respectively. The water absorption capacity of air-dried film was 5.09 g water/g dried films. For uses in the therapy of skin wounds, the potential biological mechanism of action of BC film was evaluated by using human keratinocytes and fibroblasts. Our results were the first direct demonstration that BC film supported the growth, spreading, and migration of human keratinocytes but not those of human fibroblasts. Expressions of E-cadherin and the alpha-3 chain of laminin confirmed the phenotype of human keratinocytes on BC film.

Control of culture environment for improved polyethylenimine-mediated transient production of recombinant monoclonal antibodies by CHO cells.

Abstract: In this study we describe optimization of polyethylenimine (PEI)-mediated transient production of recombinant protein by CHO cells by facile manipulation of a chemically defined culture environment to limit accumulation of nonproductive cell biomass, increase the duration of recombinant protein production from transfected plasmid DNA, and increase cell-specific production. The optimal conditions for transient transfection of suspension-adapted CHO cells using branched, 25 kDa PEI as a gene delivery vehicle were experimentally determined by production of secreted alkaline phosphatase reporter in static cultures and recombinant IgG4 monoclonal antibody (Mab) production in agitated shake flask cultures to be a DNA concentration of 1.25 microg 10(6) cells(-1) mL(-1) at a PEI nitrogen:DNA phosphate ratio of 20:1. These conditions represented the optimal compromise between PEI cytotoxicity and product yield with most efficient recombinant DNA utilization. Separately, both addition of recombinant insulin-like growth factor (LR3-IGF) and a reduction in culture temperature to 32 degrees C were found to increase product titer 2- and 3-fold, respectively. However, mild hypothermia and LR3-IGF acted synergistically to increase product titer 11-fold. Although increased product titer in the presence of LR3-IGF alone was solely a consequence of increased culture duration, a reduction in culture temperature post-transfection increased both the integral of viable cell concentration (IVC) and cell-specific Mab production rate. For cultures maintained at 32 degrees C in the presence of LR3-IGF, IVC and qMab were increased 4- and 2.5-fold, respectively. To further increase product yield from transfected DNA, the duration of transgene expression in cell populations maintained at 32 degrees C in the presence of LR3-IGF was doubled by periodic resuspension of transfected cells in fresh media, leading to a 3-fold increase in accumulated Mab titer from approximately 13 to approximately 39 mg L(-1). Under these conditions, Mab glycosylation at Asn297 remained essentially constant and similar to that of the same Mab produced by stably transfected GS-CHO cells. From these data we suggest that the efficiency of transient production processes (protein output per rDNA input) can be significantly improved using a combination of mild hypothermia and growth factor(s) to yield an extended "activated hypothermic synthesis".

A Systematic Approach for Scale-Down Model Development and Characterization of Commercial Cell Culture Processes

Abstract: The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.

Effect of membrane charge on flow and protein transport during ultrafiltration.

Abstract: Although several recent studies have demonstrated the importance of electrostatic interactions in ultrafiltration, there have been few quantitative studies of the effects of membrane charge density on protein transport and membrane hydraulic permeability. Data were obtained using a series of charge-modified cellulose membranes, with the surface charge density controlled by varying the extent of addition of a quaternary amine functionality. The membrane charge was evaluated from streaming potential measurements. Protein transmission decreased by a factor of 100 as the membrane ζ potential increased from 0.3 to 6.6 mV. The protein sieving data were in good agreement with a partitioning model accounting for electrostatic effects, while the hydraulic permeability data were consistent with a flow model accounting for the effects of counter-electroosmosis. The results provide the first quantitative analysis of the effects of membrane charge density on the performance of ultrafiltration membranes.

Optimization of fed-batch Saccharomyces cerevisiae fermentation using dynamic flux balance models

Abstract: We developed a dynamic flux balance model for fed-batch Saccharomyces cerevisiae fermentation that couples a detailed steady-state description of primary carbon metabolism with dynamic mass balances on key extracellular species. Model-based dynamic optimization is performed to determine fed-batch operating policies that maximize ethanol productivity and/or ethanol yield on glucose. The initial volume and glucose concentrations, the feed flow rate and dissolved oxygen concentration profiles, and the final batch time are treated as decision variables in the dynamic optimization problem. Optimal solutions are generated to analyze the tradeoff between maximal productivity and yield objectives. We find that for both cases the prediction of a microaerobic region is significant. The optimization results are sensitive to network model parameters for the growth associated maintenance and P/O ratio. The results of our computational study motivate continued development of dynamic flux balance models and further exploration of their application to productivity optimization in biochemical reactors.

Characterization and application of a miniature 10 mL stirred-tank bioreactor, showing scale-down equivalence with a conventional 7 L reactor

Abstract: The aim of this study was to characterize the engineering environment of an instrumented 10 mL miniature stirred-tank bioreactor and evaluate its potential as a scale-down device for microbial fermentation processes. Miniature bioreactors such as the one detailed in this work have been developed by several research groups and companies and seek to address the current bottleneck at the screening stage of bioprocess development. The miniature bioreactor was characterized in terms of overall volumetric oxygen transfer coefficient and mixing time over a wide range of impeller speeds. Power input to the miniature bioreactor was directly measured, and from this the power number of each impeller was calculated and specific power input estimated, allowing the performance of the miniature bioreactor to be directly compared with that of a conventional 7 L bioreactor. The capability of the miniature bioreactor to carry out microbial fermentations was also investigated. Replicate batch fermentations of Escherichia coli DH5alpha producing plasmid DNA were performed at equal specific power input, under fully aerobic and oxygen-limiting conditions. The results showed a high degree of equivalence between the two scales with regard to growth and product kinetics. This was underlined by the equal maximum specific growth rate and equal specific DNA product yield on biomass obtained at the two scales of operation, demonstrating the feasibility of scaling down to 10 mL on the basis of equivalent specific power input.

Enhanced secretion of heterologous proteins in Pichia pastoris following overexpression of Saccharomyces cerevisiae chaperone proteins.

Abstract: In Pichia pastoris, secretory proteins are folded and assembled in the endoplasmic reticulum (ER). However, upon introduction of foreign proteins, heterologous proteins are often retained in the cytoplasm or in the ER as a result of suboptimal folding conditions, leading to protein aggregation. The Hsp70 and Hsp40 chaperone families in the cytoplasm or in ER importantly regulate the folding and secretion of heterologous proteins. However, it is not clear which single chaperone is most important or which combination optimally cooperates in this process. In the present study we evaluated the role of the chaperones Kar2p, Sec63, YDJ1p, Ssalp, and PDI from Saccharomyces cerevisiae. We found that the introduction of Kar2p, Ssalp, or PDI improves protein secretion 4-7 times. In addition, we found that the combination chaperones of YDJlp/ PDI, YDJlp/Sec63, and Kar2p/PDI synergistically increase secretion levels 8.7, 7.6, and 6.5 times, respectively. Therefore, additional integration of chaperone genes can improve the secretory expression of the heterologous protein. Western blot experiments revealed that the chaperones partly relieved the secretion bottleneck resulting from foreign protein introduction in P. pastoris. Therefore, the findings from the present study demonstrate the presence of a network of chaperones in vivo, which may act synergistically to increase recombinant protein yields.

Cell-Free Protein Synthesis System Prepared from Insect Cells by Freeze-Thawing

Abstract: We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and beta-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5'-untranslated region (5'-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5'-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5'-UTRs tested. As a result, in a batch reaction approximately 71 microg of luciferase was synthesized per milliliter of reaction volume at 25 degrees C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5'-UTR of mRNA, approximately 45 microg/mL of luciferase was synthesized in an Sf21 cell-free system at 25 degrees C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method.

Culture of neural stem cells in calcium alginate beads.

Abstract: Neural stem cells (NSCs) with the capacity of extensive self-renewal and multilineage differentiation have attracted more and more attention in research as NSCs will play an important role in the nerve disease treatment and nerve injury repair. The shortage of NSCs, both their sources and their numbers, however, is the biggest challenge for their clinic application, and hence, in vitro culture and expansion of NSCs is vitally important to realize their potentials. In this work, mouse-derived NSCs were cultured in three-dimensional calcium alginate beads (Ca-Alg-Bs). Gelling conditions, cell density, and cell harvest were determined by the exploration of formation and dissociation parameters for Ca-Alg-Bs. Additionally, the recovered and the subsequent induced cells were identified by immunofluorescence staining of Nestin, beta-tubulin, and GFAP. The results show that the 2-mm diameter Ca-Alg-Bs, prepared with 1.5% sodium alginate solution and 3.5% CaCl2 solution and with gelling for 10 min, is suitable for the NSCs culture. The seeding density of 0.8 x 10(5) cells x mL-1 for the encapsulation of NSCs resulted in the most expansion, and the NSCs almost doubled during the experiment. The average cell recovery rate is over 88.5%, with the Ca-Alg-Bs dissolving in 55 mM sodium citrate solution for 10 min. The recovered cells cultured in the Ca-Alg-Bs still expressed Nestin and had the capacity of multilineage differentiation into neurons and glial cells and, thus, remained to be NSCs. These results demonstrate that NSC expansion within Ca-Alg-Bs is feasible and provides further possibilities for NSC expansion in bioreactors of the scale of clinical relevance.

Synthesis of novel porous magnetic silica microspheres as adsorbents for isolation of genomic DNA

Abstract: An improved procedure is described for preparation of novel mesoporous microspheres consisting of magnetic nanoparticles homogeneously dispersed in a silica matrix. The method is based on a three-step process, involving (i) formation of hematite/silica composite microspheres by urea-formaldehyde polymerization, (ii) calcination of the composite particles to remove the organic constituents, and (iii) in situ transformation of the iron oxide in the composites by hydrogen reductive reaction. The as-synthesized magnetite/silica composite microspheres were nearly monodisperse, mesoporous, and magnetizable, with as typical values an average diameter of 3.5 microm, a surface area of 250 m(2)/g, a pore size of 6.03 nm, and a saturation magnetization of 9.82 emu/g. These magnetic particles were tested as adsorbents for isolation of genomic DNA from Saccharomyces cerevisiae cells and maize kernels. The results are quite encouraging as the magnetic particle based protocols lead to the extraction of genomic DNA with satisfactory integrity, yield, and purity. Being hydrophilic in nature, the porous magnetic silica microspheres are considered a good alternative to polystyrene-based magnetic particles for use in biomedical applications where nonspecific adsorption of biomolecules is to be minimized.