bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optimized signal peptides for the development of high expressing CHO cell lines
Authors: Kober Lars , Zehe Christoph , Bode Juergen 
Published: November 2012

AbstractRecombinant biotherapeutic proteins such as monoclonal antibodies are mostly produced in Chinese hamster ovary (CHO) cells and pharmaceutical companies are interested in an appropriate platform technology for the development of large‐scale production processes. A major aim of our study was therefore to improve the secretion efficiency of a recombinant biotherapeutic antibody by optimizing signal peptides. Reporter molecules such as gaussia and vargula luciferase or secreted alkaline phosphatase are frequently used to this end. In striking contrast, we used a biotherapeutic antibody that was fused to 16 different signal peptides during our study. In this way, the secretion efficiency of the recombinant antibody has been analyzed by transient expression experiments in CHO cell lines. Compared to the control signal peptide, it was not possible to achieve higher efficiencies with signal peptides derived from a variety of species or even natural immunoglobulin G signal peptides. The best results were obtained with natural signal peptides derived from human albumin and human azurocidin. These results were confirmed by fed‐batch experiments with stably transfected cell pools, in which cell‐specific productivities up to 90 pg cell−1 day−1 and product concentrations up to 4 g L−1 could be determined using the albumin signal peptide. Finally, the applicability of the identified signal peptides for both different antibodies and non‐antibody products was demonstrated by transient expression experiments. In conclusion, it was found that signal peptides derived from human albumin and human azurocidin are most appropriate to generate cell lines with clearly improved production rates suitable for commercial purposes in a product‐independent manner. Biotechnol. Bioeng. 2013; 110: 1164–1173. © 2012 Wiley Periodicals, Inc.
In silico multi‐scale model of transport and dynamic seeding in a bone tissue engineering perfusion bioreactor
Authors: Spencer T.J. , Hidalgo‐Bastida L.A. , Cartmell S.H. , Halliday I. , Care C.M. 
Published: November 2012

AbstractComputer simulations can potentially be used to design, predict, and inform properties for tissue engineering perfusion bioreactors. In this work, we investigate the flow properties that result from a particular poly‐L‐lactide porous scaffold and a particular choice of perfusion bioreactor vessel design used in bone tissue engineering. We also propose a model to investigate the dynamic seeding properties such as the homogeneity (or lack of) of the cellular distribution within the scaffold of the perfusion bioreactor: a pre‐requisite for the subsequent successful uniform growth of a viable bone tissue engineered construct. Flows inside geometrically complex scaffolds have been investigated previously and results shown at these pore scales. Here, it is our aim to show accurately that through the use of modern high performance computers that the bioreactor device scale that encloses a scaffold can affect the flows and stresses within the pores throughout the scaffold which has implications for bioreactor design, control, and use. Central to this work is that the boundary conditions are derived from micro computed tomography scans of both a device chamber and scaffold in order to avoid generalizations and uncertainties. Dynamic seeding methods have also been shown to provide certain advantages over static seeding methods. We propose here a novel coupled model for dynamic seeding accounting for flow, species mass transport and cell advection‐diffusion‐attachment tuned for bone tissue engineering. The model highlights the timescale differences between different species suggesting that traditional homogeneous porous flow models of transport must be applied with caution to perfusion bioreactors. Our in silico data illustrate the extent to which these experiments have the potential to contribute to future design and development of large‐scale bioreactors. Biotechnol. Bioeng. 2013; 110: 1221–1230. © 2012 Wiley Periodicals, Inc.
Polymer characterization and optimization of conditions for the enhanced bioproduction of benzaldehyde by Pichia pastoris in a two‐ ;phase partitioning bioreactor
Authors: Craig Tom , Daugulis Andrew J. 
Published: November 2012

AbstractBenzaldehyde, with its apricot and almond‐like aroma, is the second most abundantly used molecule in the flavor industry, and is most commonly produced via chemical routes, such as by the oxidation of toluene. Biologically produced benzaldehyde, whether by extraction of plant material or via microbial biotransformation, commands a substantial price advantage, and greater consumer acceptance. Methylotrophic yeast, such as Pichia pastoris, contain the enzyme alcohol oxidase (AOX), which, in the presence of alcohols other than methanol, are able to yield aldehydes as dead‐end products, for example, benzaldehyde from benzyl alcohol. In this work, we have determined that benzaldehyde, and not benzyl alcohol, is inhibitory to the transformation reaction by P. pastoris, prompting the development of a selection strategy for identifying sequestering polymers for use in a partitioning bioreactor that was based on the ratio of partition coefficients (PCs) for the two target molecules. Additionally, we have now confirmed for the first time, that the mechanism of solute uptake by amorphous polymers is via absorption, not adsorption. Finally, we have adopted a common strategy used for the production of heterologous proteins by P. pastoris, namely the use of a mixed methanol/glycerol feed for inducing the required AOX enzyme, while reducing the time required for high density biomass generation. All of these components were combined in a final experiment in which 10% of the polymer Kraton D1102K, whose PC ratio of benzaldehyde to benzyl alcohol was 14.9, was used to detoxify the biotransformation in a 5 L partitioning bioreactor, resulting in a 3.4‐fold increase in benzaldehyde produced (14.4 g vs. 4.2 g) relative to single phase operation, at more than double the volumetric productivity (97 mg L−1 h−1 vs. 41 mg L−1 h−1). Biotechnol. Bioeng. 2013; 110: 1098–1105. © 2012 Wiley Periodicals, Inc.
Model‐based analysis on the extractability of information from data in dynamic fed‐batch experiments
Authors: Wechselberger Patrick , Sagmeister Patrick , Herwig Christoph 
Published: November 2012

AbstractDynamic changes of physiological bioprocess parameters, e.g. a change in the specific growth rate μ, are frequently observed during industrial manufacturing as well as bioprocess development. A quantitative description of these variations is of great interest, since it can bring elucidation to the physiological state of the culture. The goal of this contribution was to show limitations and issues for the calculation of rates with regard to temporal resolution for dynamic fed‐batch experiments. The impact of measurement errors, temporal resolution and the physiological activity on the signal to noise ratio (SNR) of the calculated rates was evaluated using an in‐silico approach. To make use of that in practice, a generally applicable rule of thumb equation for the estimation of the SNR of specific rates was presented. The SNR calculated by this rule of thumb equation helps with definition of sampling intervals and making a decision whether an observed change is statistically significant or should be attributed to random error. Furthermore, a generic reconciliation approach to remove random as well as systematic error from data was presented. This reconciliation technique requires only little prior knowledge. The validity of the proposed tools was checked with real data from a fed‐batch culture of E. coli with dynamic variations due to feed profile. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013
Recombinant expression and purification of the antimicrobial peptide magainin‐2
Authors: Ramos Reinaldo , Moreira Susana , Rodrigues Ana , Gama Miguel , Domingues Lucília 
Published: November 2012

AbstractMagainin‐2 (MAG2) is a polycationic antimicrobial peptide isolated from the skin of the African clawed frog Xenopus laevis. It has a wide spectrum of antimicrobial activities against gram‐positive and gram‐negative bacteria, fungi, and induces osmotic lysis of protozoa. MAG2 also possesses antiviral and antitumoral properties. These activities make this peptide a promising candidate for therapeutic applications. Recombinant expression systems are necessary for the affordable production of large amounts of the biologically active peptide. In this work, MAG2 has been cloned to the N‐terminal of a family III carbohydrate‐binding module fused to the linker sequence (LK‐CBM3) from Clostridium thermocellum; a formic acid recognition site was introduced between the two modules for chemical cleavage of the peptide. The recombinant protein MAG2‐LK‐CBM3 was expressed in Escherichia coli BL21 (DE3) and MAG2 was successfully cleaved and purified from the fusion partner LK‐CBM3. Its functionality was confirmed by testing its activity against gram‐negative bacteria. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines
Authors: Rodríguez‐Nogales José Manuel , Vila‐Crespo Josefina , Fernández‐Fernández Encarnación 
Published: November 2012

AbstractEntrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell‐loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell‐loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
Avidity‐mediated virus separation using a hyperthermophilic affinity ligand
Authors: Hussain Mahmud , Lockney Dustin , Wang Ruqi , Gera Nimish , Rao Balaji M. 
Published: November 2012

AbstractImmunoaffinity separation of large multivalent species such as viruses is limited by the stringent elution conditions necessary to overcome their strong and highly avid interaction with immobilized affinity ligands on the capture surface. Here we present an alternate strategy that harnesses the avidity effect to overcome this limitation. Red clover necrotic mosaic virus (RCNMV), a plant virus relevant to drug delivery applications, was chosen as a model target for this study. An RCNMV binding protein (RBP) with modest binding affinity (KD ∼100 nM) was generated through mutagenesis of the Sso7d protein from Sulfolobus solfataricus and used as the affinity ligand. In our separation scheme, RCNMV is captured by a highly avid interaction with RBP immobilized on a nickel surface through a hexahistidine (6xHis) tag. Subsequently, disruption of the multivalent interaction and release of RCNMV is achieved by elution of RBP from the nickel surface. Finally, RCNMV is separated from RBP by exploiting the large difference in their molecular weights (∼8 MDa vs. ∼10 kDa). Our strategy not only eliminates the need for harsh elution conditions, but also bypasses chemical conjugation of the affinity ligand to the capture surface. Stable non‐antibody affinity ligands to a wide spectrum of targets can be generated through mutagenesis of Sso7d and other hyperthermophilic proteins. Therefore, our approach may be broadly relevant to cases where capture of large multivalent species from complex mixtures and subsequent release without the use of harsh elution conditions is necessary. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
Fed‐batch CHO cell t‐PA production and feed glutamine replacement to reduce ammonia production
Authors: Kim Do Yun , Chaudhry Muhammad Arshad , Kennard Malcolm L. , Jardon Mario A. , Braasch Katrin , Dionne Ben , Butler Michael , Piret James M. 
Published: November 2012

AbstractIndustrial therapeutic protein production has been greatly improved through fed‐batch development. In this study, improvement to the productivity of a tissue‐plasminogen activator (t‐PA) expressing Chinese hamster ovary (CHO) cell line was investigated in shake flask culture through the optimization of the fed‐batch feed and the reduction of ammonia generation by glutamine replacement. The t‐PA titer was increased from 33 mg/L under batch conditions to 250 mg/L with daily feeding starting after three days of culture. A commercially available fed‐batch feed was supplemented with cotton seed hydrolysate and the four depleted amino acids, aspartic acid, asparagine, cysteine, and tyrosine. The fed‐batch operation increased the generation of by‐products such as lactate and ammonia that can adversely affect the fed‐batch performance. To reduce the ammonia production, a glutamine‐containing dipeptide, pyruvate, glutamate, and wheat gluten hydrolysate, were investigated as glutamine substitutes. To minimize the lag phase as the cells adjusted to the new energy source, a feed glutamine replacement process was developed where the cells were initially cultured with a glutamine containing basal medium to establish cell growth followed by feeding with a feed containing the glutamine substitutes. This two‐step feed glutamine replacement process not only reduced the ammonia levels by over 45% but, in the case of using wheat gluten hydrolysate, almost doubled the t‐PA titer to over 420 mg/L without compromising the t‐PA product quality or glycosylation pattern. The feed glutamine replacement process combined with optimizing other feed medium components provided a simple, practical, and effective fed‐batch strategy that could be applied to the production of other recombinant therapeutic proteins. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
Solubility properties and diffusional extraction behavior of natamycin from streptomyces gilvosporeus biomass
Authors: Zeng Xianhai , Danquah Michael K. , Jing Keju , Woo Meng Wai , Chen Xiao Dong , Xie Youping , Lu Yinghua 
Published: November 2012

AbstractNatamycin is a type of polyene macrolide antibiotic and has been produced in submerged microbial cultures of some natural Streptomyces strains. Natamycin extraction from cellular biomass is greatly affected by the molecular and solubilization characteristics of the extraction solvent, and this is a major reason for the routine attainment of low volumetric titers, resulting from sparing natamycin solubility. In this work, a series of experiments were conducted to investigate the solubility of natamycin in some selected organic solvents in order to assess the influence on natamycin extraction yield. Natamycin showed the highest solubility in 75% aqueous methanol under the conditions of pH 2, 30°C and 1 atm. Furthermore, the extraction of natamycin using 75% aqueous methanol was performed and the highest extraction yield of 45.7% was obtained under pH 2. A mathematical model derived from Fick's law of the biomolecular diffusion process was developed to fit the experimental kinetic data of natamycin extraction. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
Optimization of a two‐step process comprising lipase catalysis and thermal cyclization improves the efficiency of synthesis of six‐membered cyclic carbonate from trimethylolpropane and dimethylcarbonate
Authors: Bornadel Amin , Hatti‐Kaul Rajni , Sörensen Kent , Lundmark Stefan , Pyo Sang‐Hyun 
Published: November 2012

AbstractSix‐membered cyclic carbonates are potential monomers for phosgene and/or isocyanate free polycarbonates and polyurethanes via ring‐opening polymerization. A two‐step process for their synthesis comprising lipase‐catalyzed transesterification of a polyol, trimethylolpropane (TMP) with dimethylcarbonate (DMC) in a solvent‐free system followed by thermal cyclization was optimized to improve process efficiency and selectivity. Using full factorial designed experiments and partial least squares (PLS) modeling for the reaction catalyzed by Novozym®435 (N435; immobilized Candida antarctica lipase B), the optimum conditions for obtaining either high proportion of monocarbonated TMP and TMP‐cyclic‐carbonate (3 and 4), or dicarbonated TMP and monocarbonated TMP‐cyclic‐carbonate (5 and 6) were found. The PLS model predicted that the reactions using 15%–20% (w/w) N435 at DMC:TMP molar ratio of 10–30 can reach about 65% total yield of 3 and 4 within 10 h, and 65%–70% total yield of 5 and 6 within 32–37 h, respectively. High consistency between the predicted results and empirical data was shown with 66.1% yield of 3 and 4 at 7 h and 67.4% yield of 5 and 6 at 35 h, using 18% (w/w) biocatalyst and DMC:TMP molar ratio of 20. Thermal cyclization of the product from 7 h reaction, at 110°C in the presence of acetonitrile increased the overall yield of cyclic carbonate 4 from about 2% to more than 75% within 24 h. N435 was reused for five consecutive batches, 10 h each, to give 3+4 with a yield of about 65% in each run. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
Osteogenic differentiation and osteochondral tissue engineering using human adipose‐derived stem cells
Authors: Mahmoudifar Nastaran , Doran Pauline M. 
Published: November 2012

AbstractOsteogenesis and the production of composite osteochondral tissues were investigated using human adult adipose‐derived stem cells and polyglycolic acid (PGA) mesh scaffolds under dynamic culture conditions. For osteogenesis, cells were expanded with or without osteoinduction factors and cultured in control or osteogenic medium for 2 weeks. Osteogenic medium enhanced osteopontin and osteocalcin gene expression when applied after but not during cell expansion. Osteogenesis was induced and mineralized deposits were present in tissues produced using PGA culture in osteogenic medium. For development of osteochondral constructs, scaffolds seeded with stem cells were precultured in either chondrogenic or osteogenic medium, sutured together, and cultured in dual‐chamber stirred bioreactors containing chondrogenic and osteogenic media in separate compartments. After 2 weeks, total collagen synthesis was 2.1‐fold greater in the chondroinduced sections of the composite tissues compared with the osteoinduced sections; differentiation markers for cartilage and bone were produced in both sections of the constructs. The results from the dual‐chamber bioreactor highlight the challenges associated with achieving simultaneous chondrogenic and osteogenic differentiation in tissue engineering applications using a single stem‐cell source. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013
Determination of Heat Treatment Temperature for Completing Polycondensation of Vinyl Substituted Silica Particles Prepared by Sol‐Gel Method
Authors: Ashraf K. M. , Matsumoto S. , Kurumada K. 
Published: November 2012

AbstractThe completion of the polycondensation of sol‐gel derived organic‐inorganic hybrid silica by heat treatment was studied for powder samples prepared from vinyltrimethoxysilane and vinyltriethoxysilane. The appropriate temperature for the completion of the prepared submicron powdery samples from both starting compounds was determined through thermogravimetric analysis. The completion or incompletion of the polycondensation was elucidated by means of several instrumental methods in addition to a light scattering test. The lowest heat treatment conditions for the particles from vinyltrimethoxysilane and vinyltriethoxysilane are 170 °C and 180 °C, respectively, for 24 h. The morphologies of the heat treated samples were found to be slightly different depending on the starting material used.
Preparation of Agy zeolites using microwave irradiation and study on their adsorptive desulphurisation performance
Authors: Song Hua , Wan Xia , Sun Xinglong 
Published: November 2012

AbstractSorbents with Ag loading supported on NaY were synthesised by a liquid‐phase ion exchange method taking advantage of microwaves irradiation. The adsorption desulphurisation activity of the AgY adsorbent exchanged with AgNO3 solution of 0.2 mol L−1, microwave irradiation time 20 min with power 240 W as well as calcined at 500°C exhibited the best performance. Equilibrium isotherm shows that the adsorption of thiophene over the as‐prepared AgY can be represented by the Langmuir model. Kinetics of adsorptive desulphurisation followed the pesudo‐second‐order model and the equilibrium adsorption capacity q2 = 0.893 mmol g−1. © 2012 Canadian Society for Chemical Engineering
New algebraic expressions for thermodiffusion in binary n‐alkane mixtures
Authors: Srinivasan Seshasai , Saghir M. Ziad 
Published: November 2012

AbstractFollowing a detailed analysis of the experimental data on thermodiffusion coefficients of numerous binary hydrocarbon mixtures from over 90 experiments, four new algebraic expressions are proposed. As per these formulations, the thermodiffusion coefficient of binary n‐alkane mixtures can be expressed as a function of the chemical composition of the mixture and the mixture properties such as density and viscosity. Detailed experimental validation is presented using four n‐alkane series containing a wide range of combinations of n‐alkanes. Additionally, comparison with recently proposed neural network model and a model based on the principles of non‐equilibrium thermodynamics is also presented. It has been found that the proposed algebraic models are simple in formulations, are evaluated with least computational effort and yet have a very high accuracy in predicting the thermodiffusion coefficients. © 2012 Canadian Society for Chemical Engineering
The effect of poly(ethylene‐alt‐maleic anhydride) on the reduction crystallisation behaviour of nickel powder
Authors: Bodoza T.F. , Ntuli F. , Chauke D.F. , Muzenda E. 
Published: November 2012

AbstractThe effect of poly(ethylene‐alt‐maleic anhydride) (EMA), an additive used in nickel powder precipitation, has been studied using sodium hypophosphite as a reducing agent. Reduction experiments were conducted using a 10 L stainless steel batch reactor. The effect of EMA was investigated by studying the evolution of the particle size distribution (PSD) and its derived moments, specific surface area, rate of reduction, pH–redox potential and elemental composition of the powder product. EMA has been found to act as a reduction catalyst and anti‐agglomerating agent. The major particulate processes identified were size dependent aggregation, molecular growth and breakage. © 2012 Canadian Society for Chemical Engineering