bluuebunny/crossref_metadata_2025 huggingface dataset:
Host cell protein clearance during protein a chromatography: Development of an improved column wash step
Authors: Shukla Abhinav A. , Hinckley Peter
Published: December 2008

Host cell protein (HCP) contaminant clearance is a significant concern during downstream process development for biopharmaceuticals. Protein A chromatography as a capture step for monoclonal antibodies and Fc fusion proteins can clear a large proportion of these impurities from cell culture harvest. Nevertheless, remaining levels of this process‐related impurity class do present significant constraints on the rapid development of effective and robust polishing steps. Conventionally, an intermediate pH wash is employed between column loading and elution to minimize HCP levels after Protein A chromatography. A significant mechanistic finding presented in this work is that HCP contaminants that persist following Protein A capture predominantly comprise species that associate with the product in preference to direct interaction with the chromatographic resin. This suggests that the development of improved column wash techniques to maximize HCP clearance ought to focus on disrupting protein–HCP interactions rather than Protein A–HCP interactions. A higher wash pH to preserve product – Protein A binding along with the use of additive combinations to disrupt interactions between HCPs and the product are investigated. This strategy was successfully applied to develop a broadly applicable wash condition that has the potential for eliminating the need for product specific optimization of wash conditions. A combination of 1 M urea and 10% isopropanol in the wash buffer were successfully applied as a platform wash condition for Protein A chromatography. Use of this (and other similar) wash conditions are anticipated to aid the rapid development of effective downstream processes for monoclonal antibodies and Fc fusion proteins resulting in their rapid introduction into clinical trials.
Enhancing cell affinity of nonadhesive hydrogel substrate: The role of silica hybridization
Authors: Wang Chunming , Bai Jing , Gong Yihong , Zhang Feng , Shen Jiangbo , Wang Dong‐An
Published: December 2008

The aim of this study is to elucidate the functional mechanism of nanosilica‐induced cell affinity for the normally attachment‐fouling hydrogel substrates. Investigations were conducted in the following three major aspects: (1) environmental protein adsorption before cell contacts, which determines the presence of adhesive ligands; (2) integrin expression profile, which reflects the cell responses via surface receptor turning‐over; and (3) vinculin activation and F‐actin assembly, which sketches the induced cytoskeletal organization posing for focal adhesion. The results indicate that the hybridized nanosilica additives are capable of arresting adhesive proteins from the environment. As binding ligands, such immobilized proteins activate α5β1‐specific pathway for cell focal adhesion, but fail in invoking β3‐participated signaling cascade. This partial activation enables modest amount of high‐quality cell adhesion on the modified substrate, by which a conservative cell affinity is established on hydrogel matrices.
Separation of protein charge variants with induced pH gradients using anion exchange chromatographic columns
Authors: Pabst Timothy M. , Carta Giorgio , Ramasubramanyan Natrajan , Hunter Alan K. , Mensah Paul , Gustafson Mark E.
Published: December 2008

This work concerns the chromatographic separation of protein charge variants using pH gradients generated by step changes in buffer composition with weak base anion exchange columns. A local equilibrium model is first developed to describe pH transitions occurring in the column using buffers comprising neutral, zwitterionic or positively charged species. Model predictions, based solely on the resins' titration curves and obtained with the method of characteristics, show, in excellent agreement with experiments, that induced pH gradients of varying durations and shapes can be obtained with a broad range of buffer systems including Tris, Bis‐Tris propane, histidine, and their mixtures and ethanolamine. The separation of protein charge variants is then demonstrated for bovine apo‐transferrin and for a monoclonal antibody. The resolution of the charge variants present in these proteins, demonstrated via isoelectric focusing analyses, is obtained for conditions amenable to scale‐up for preparative purposes; that is larger particle sizes (90 μm), higher flow rates (100–600 cm/h), and higher protein loads (2–5 mg/mL). Because the approach requires only step changes in buffer composition and commonly available, unretained buffers species, practical implementation is straightforward. The focusing effect of the induced pH gradient results in relatively sharp peaks and substantial resolution even for these conditions.
High Resolution Continuous Flow Electrophoresis
Authors: Gobie William A. , Beckwith James B. , Ivory Cornelius F.
Published: December 2008

Continuous free‐flow electrophoresis (CFE) provides the biochemist with a powerful technique for preparative‐scale purification of labile biomolecules. However, solute resolution in the classic CFE is limited by the deleterious effects of diffusion, electroosmotic dispersion, and connective dispersion. These problems can be overcome in the CFE by continuously recycling effluent through the chamber in a manner which allows complete recovery of undiluted product at arbitrarily high purities.

Mathematical models of the continuous flow device with recycle (RCFE) are derived for the two limiting cases: rapid diffusion and slow diffusion. The analytical solutions of these models are used to predict separation factors under various operating conditions.
Protein Extraction Using Reverse Micelles
Authors: Cöklen Kent E. , Hatton T. Alan
Published: December 2008

The controlled solubilization of proteins in a reverse micelle‐containing organic phase is considered as a method for separation of proteins by liquid‐liquid extraction. Cytochrome‐C was transferred between a bulk aqueous phase and a micellar phase, using the Aerosol OT‐isooctane system of surfactant and solvent. It was observed that the extent of protein solubilization could be controlled by varying the ionic strength of the aqueous phase equilibrated with the micellar phase.

The protein transferred into the micellar phase rapidly, under conditions of low ionic strength, and transferred out of the micelles relatively slowly, under conditions of high ionic strength. It is proposed that the effect of the ionic strength was to alter the electrostatic interaction between the surface charge of the protein and the charged interior of the reverse micelle by Debye screening.
Continuous Immobilized Recombinant Protein Production from E. coli Capable of Selective Protein Excretion: A Feasibility Study
Authors: Georgiou G. , Chalmers J. J. , Shuler M. L. , Wilson D. B.
Published: December 2008

Currently genetically engineered bacteria are economically attractive only for the production of high‐value, low‐volume‐proteins. For this technology to be useful for the manufacture of high volume proteins new bioprocessing concepts are necessary. A potential approach is to construct a plasmid containing an inducible promoter and a hybrid gene containing a signal sequence fused to the gene for the protein to be produced. The plasmid is transformed into Escherichia coli. The plasmid‐host interaction is such that the host becomes “leaky” upon introduction. Since the signal sequence allows the recombinant protein to pass the cytoplasmic membrane, a “leaky cell” can allow selective excretion of the desired protein. If the “leaky” cell is immobilized and maintained in a resting state, the sustained continuous production of the target protein is possible. To test this concept we have used plasmid pKK in E. coli RB791 with β‐lactamase as the target protein. Production levels of at least 390 units β‐lactamase/L‐h can be sustained for at least 120 days. Peak productivities of 10,000 unitslL‐h have been obtained. The β‐lactamase constitutes approximately 40% of the protein in solution. Such systems could reduce recombinant protein production costs due both to the continuous nature of the bioreactor and the improved ease of protein recovery.
Continuous Affinity Chromatography Using a Magnetically Stabilized Fluidized Bed
Authors: Burns Mark A. , Graves David J.
Published: December 2008

A new processing tool, the magnetically stabilized fluidized bed, offers opportunities for efficient fluid‐solids contact.

The bubbling and mixing normally seen in a fluidized bed are eliminated, and continuous countercurrent contact is possible. This technique is especially suitable for biochemical separations, where cellular debris would clog an ordinary fixed bed. Preliminary modeling and experimental work on such a system is discussed, along with details of a relatively simple experimental system.
Aerosol Formation During the Microwave Freeze Dehydration of Beef
Authors: Arsem H. B. , Ma Y. H.
Published: December 2008

Data are obtained from the combined microwave and radiant freeze drying of beef steaks. Causes for the high drying rates are explained by the entrainment of solid ice crystals in the vapor during the low pressure drying. The effects of solid entrainment on dehydration rate and product quality are discussed in terms of the loss of intercellular salts and proteins and tearing of muscle fibers caused by high vapor velocity.
Flow Cytometry Analysis of Plasmid Amplification in Escherichia coli
Authors: Seo Jin‐Ho , Srienc Friedrich , Bailey James E.
Published: December 2008

Large quantities of cloned DNAs for use in characterization, or in subsequent recombinant plasmid construction, may be obtained by plasmid amplification, which is the increase of cellular plasmid DNA content relative to the amount of host cell chromosomal DNA. Plasmid amplification has been investigated with flow cytometry in chloramphenicol (Cm) treated cell populations of Escherichia coli HB101 carrying plasmids with pMBI replication origins at different copy number levels ranging from 12 to 122. The flow cytometry method is based on the determination of the total cellular DNA content after staining with mithramycin, a DNA‐specific fluororescent dye. This method permits direct measurement of the rate of plasmid amplification and classification of cell populations according to chromosomal replication forks at the time of Cm treatment. Both initial rates of plasmid amplification and final contents of amplified plasmids were found to be proportional to the average initial plasmid copy numbers. The maximum degree of amplification, defined as the ratio of plasmid content after eighteen hours incubation to the preamplification plasmid content at steady‐state growth, was approximately equal for the strains studied.

Larger cells, with four active chromosomal replication forks at Cm addition, exhibit faster plasmid replication rates than cells initially at earlier cell cycle states. A kinetic model was proposed that agrees well with the present and previous experimental observations. This work clearly demonstrates the advantage of flow cytometry for experimental analysis of plasmid propagation in recombinant organisms.
Simplified Models for Estimating Isothermal Operating Characteristics of Food Extruders
Authors: Levine Leon , Rockwood Jeffrey
Published: December 2008

A model of isothermal food extruder performance is described. Inferences about alternative extruder screw designs and their performance are drawn from the model. The model suggests that thread depth or diameter compression screws are superior in performance to a pitch compression screw. The advantage gained from using diameter compression screws is paid for with significantly higher rates of energy dissipation. The use of the model to characterize screws having both a compression zone and metering zone is described.
Microbial Desulfurization of Coals in a Slurry Pipeline Reactor Using. Thiobacillus ferrooxidans
Authors: Rai Charanjit
Published: December 2008

Microbial desulfurization of Illinois #6 and Indiana #3 bituminous coal having a total sulfur content of 2 to 8% has been investigated using acidophilic microorganism, Thiobacillus ferrooxidans, in laboratory shake flash experiments and in a two‐inch pipeline loop. The results indicate that about 80 to 85% pyritic sulfur removal was achieved with 10 to 25% coal/water slurry recirculated at 6–7 ft/sec at room temperature in 7 to 14 days. The experimental conditions have been optimized for maximum desulfurization. Results from this study show that the rates of bacterial desulfurization from coal samples are higher in the pipeline loop under turbulent flow conditions as compared to the shake‐flask experiments for particle sizes ranging from 43 to 200 μm. It is visualized that the proposed coal slurry pipelines could be used as biological plug flow reactors under aerobic conditions. The laboratory corrosion studies under dynamic test conditions show that use of a corrosion inhibitor will limit the pipeline corrosion rate to acceptable levels.
The Relationship Between Cell Dry Weight Concentration and Culture Turbidity For A Recombinant E. Coli K12 Strain Producing High Levels of Human Alpha Interferon Analogue
Authors: Fieschko John , Ritch Tom , Bengston David , Fenton Dennis , Mann Michael
Published: December 2008

A recombinant E. Coli K12 strain carrying a runaway‐type plasmid with the gene for human alpha interferon analogue and capable of producing high levels of recombinant product [1], was grown in rich medium and compared to the host strain without plasmid, and to the strain with the plasmid, but lacking the interferon gene insert. At a non‐inducing temperature, gross cell morphology and the correlation between dry weight and culture turbidity was comparable for the three cultures. Following induction by temperature elevation, both plasmid‐bearing strains displayed significant cell size change, but only the interferon producing strain underwent a decrease in the ratio of cell concentration measured by dry weight to culture turbidity.
Modeling the Stability of Cephamycin C—Producing N. Lactamdurans During Continuous Culture
Authors: Kirpekar A. C. , Kirwan D. J. , Stieber R. W.
Published: December 2008

The reversion of a cephamycin C‐producing strain of Nocardia lactamdurans to a non‐producing variant under continuous culture conditions was examined at dilution rates between 0.025 and 0.045 hr−1. A model incorporating the influence of revertants, when present in the culture in significant numbers, was necessary to adequately describe the dynamics of the reversion process.
Evaluation of Rotational Flow Stabilized Continuous Electrophoresis for Protein Fractionation
Authors: Noble Paul T.
Published: December 2008

A research group at Harwell Laboratory have invented a relatively large‐scale electrophoretic separater, which uses angular momentum to stabilize the fluid flow against thermally induced mixing. An earlier review describes the device and shows potential industrial application. Additional experiments and modeling are reported here to help clarify its potential for industrial protein fractionation. The scale‐up potential of the flow stabilization is not considered.
A Novel Bioreactor‐Cell Precipitator Combination for High‐Cell Density, High‐Flow Fermentations
Authors: Stephanopoulos Gregory , San K.‐Y. , Davison B. H.
Published: December 2008

A novel bioreactor design has been investigated for carrying out in a single unit a fermentation and cell recycle process simultaneously. The reactor consisted of a typical fermentor and a side‐arm attached at a certain fixed angle to the fermentor. Due to the enhanced sedimentation in the inclined side‐arm, the cells precipitate quickly and flow back into the fermentor. This allowed an essentially cell‐free fermentation broth to be withdrawn at high rates through the sidearm while maintaining a high cell density culture in the fermentor. Continuous flow fermentation runs of S. cerevisiae demonstrated these features and showed that many fold increases in the steady‐state cell density could be achieved using high flowrates, which in an ordinary chemostat would lead to washout. Ethanol productivities were high and can further be increased several‐fold through a straightforward scale‐up of the precipitator sidearm to sustain even larger flow rates. Discussed also are various other reactor features such as the enhanced resistance to contamination, possible reduction of the plasmid instability problems of recombinant microorganisms, potential applications with flocculent strains, steady‐state coexistence of competing populations in mixed culture, and the possibility of carrying out fermentations with stationary cultures under continuous flow conditions.
Stable Competitive Coexistence in a Continuous Fermentor with Size‐Selective Properties
Authors: Davison B. H. , San K.‐Y. , Stephanopoulos Gregory
Published: December 2008

A reactor with size‐selective properties was found to allow a mixed culture of Escherichia coli and Saccharomyces cerevisiae to coexist under continuous culture conditions. The reactor consisted of a fermentor with an attached inclined side‐arm to recycle the cells by enhanced sedimentation. The larger size yeast population was retained preferentially and recycled at high efficiencies while the smaller yet faster growing bacteria were removed through the side‐arm. Stability analysis indicated that the coexistence of this system could be stable only if the net yeast removal rate through the side‐arm as a function of the reactor yeast concentration was concave up. This would occur if growth continued in the side‐arm or if settling efficiency decreased at higher cell densities. Another experimental system was devised to measure this net removal rate function. A negative removal rate (i.e., a net addition of yeast to the fermentor) was observed at low biomass indicating growth in the settler and explaining the stability of the coexistence steady state.
An Application of the Method of Lines in Biological Mass Transfer
Authors: Mayes Peter J. D.
Published: December 2008

The method of lines is a numerical technique particularly suited to the solution of parabolic partial differential equations, which has been the subject of much research in recent years, and which has been incorporated into a number of readily available computer library packages. The method has been applied to a number of problems in areas such as chemical engineering, atmospheric physics and nuclear engineering. It does not however, seem to have been widely used in biological mass transfer problems of the type arising in the study of artificial lungs and kidneys. By describing some of the problems tackled by the author and his colleagues, it is hoped to interest a wider audience in the potential benefits of this method.
Solubilization of Biomolecules in Carbon Dioxide Based Supercritical Fluids
Authors: Wong J. M. , Johnston K. P.
Published: December 2008

Supercritical fluid (SF) carbon dioxide offers several advantages compared with organic liquid solvents for separations and reaction processes of thermally labile biomolecules.

A major limitation is that the even moderately polar biomolecules are only slightly soluble. Experimental solubility and vapor pressure data were obtained for cholesterol, stigmasterol, and ergosterol in SF CO2 with and without co‐solvents over a pressure range of 100 to 350 bar. Small concentrations of certain co‐solvents can increase solubilities of particular sterols by one or two orders of magnitude, but have little effect on other sterols due to complexes formed in the solid phase. The experimental data were correlated using component solubility parameters to obtain the unlike‐pair attraction constant used in a modified van der Waals equation of state.
Ethylene From Penicillium Digitatum
Authors: Tanyolac Abdurrahman M. , Gainer John L.
Published: December 2008

Ethylene is an example of a bulk chemical commodity that could be produced by fermentation. The production of ethylene using Penicillium digitatum, and the effects of adapting this fungus to a submerged culture, are reported here.
Multimembrane Bioreactor For Extractive Fermentation
Authors: Cho T. , Shuler M. L.
Published: December 2008

A multimembrane reactor is described. Four layers (gas, cells, nutrient, and solvent) are separated by membranes. This structure prevents solvent emulsification in the fermentation broth. The system was tested for ethyl alcohol production from glucose using yeast. Tributyl phosphate (TBP) was chosen as the extractant. Experiments demonstrate for the first time a successful extractive fermentation with a practical solvent. Prevention of emulsification removes the toxic effect of TBP on yeast metabolism.
High Capacity Aeration in a Very Tall Tank
Authors: Jackson M. L.
Published: December 2008

A tall tank (13 m high by 1.8 m diameter) with four orifice air inlets showed linear relationships among air input rate, gas phase holdup, and volumetric oxygen transfer coefficient. The established performance of the tall tank aeration system may be used for reference in design when scaling from laboratory to industrial size equipment.
Electrochemical Sensors in the Analysis and Control of Bioprocesses
Authors: Twork John V. , Yacynych Alexander M.
Published: December 2008

The need for real‐time, in‐situ monitoring and control of the chemical composition of fermentation reactions has led to several novel approaches in the development of amperometric electrochemical sensors.
Evaluation of Supercritical Fluid Extraction in the Pharmaceutical Industry
Authors: Larson Karen A. , King Michael L.
Published: December 2008

Various steroids precipitate from supercritical CO2 as very small particles but still retain their crystallinity. This suggests that SCFE in this industry could be
utilized as a size reduction method as an alternative to milling. The application
of SCFE as a separation technique is demonstrated by the simultaneous extraction and reaction of mevinolin, a metabolite of the fungus Aspergillus terreus.
Simulation of Secondary Metabolite Production By Immobilized Living Cells: Penicillin Production
Authors: Okita W. B. , Kirwan D. J.
Published: December 2008

The production of secondary metabolites by immobilized living microorganisms was investigated by simulation techniques. The behavior of free cell and immobilized cell reactors for the production of penicillin by P. chrysogenum was compared. This system was chosen as a model system because literature data on growth and production kinetics are available. Simulation results for bioreactors containing P. chrysogenum immobilized by adsorptive techniques demonstrates a potential advantage in productivity for such reactors over conventional fed batch operation. Realization of this advantage requires an understanding of growth and product formation kinetics for immobilized cells as well as development of immobilization technology that will provide stable, high cell loadings.
Hemoglobin‐based oxygen carrier enhanced tumor oxygenation: A novel strategy for cancer therapy
Authors: Gundersen Sharon I. , Palmer Andre F.
Published: December 2008

A tumor's low oxygen tension is a major obstacle to current chemo‐ and radio‐therapies. By increasing oxygen transport to hypoxic tumor regions it is possible to make tumors more susceptible to current cancer treatment strategies. This article theoretically investigates the possibility of supplementing human blood with hemoglobin‐based oxygen carriers (HBOCs) in an attempt to target oxygen delivery specifically to the low oxygen tension regions present in tumors. In this work, we develop a mathematical model that describes oxygen transport in a cylindrical annulus of cancerous tissue fed by a single cylindrical capillary. The oxygen transport model was used to evaluate mixtures of red blood cells and six types of HBOCs that consisted of two polymerized hemoglobins, two liposome‐encapsulated hemoglobins, and two hydrogel‐encapsulated hemoglobins. These HBOCs span a wide range of oxygen affinities (P50s) ranging from 5 to 54 mm Hg to study the effect of P50 on the ability to target oxygen delivery to hypoxic regions of a tumor and hence improve tumor oxygenation. The results of these simulations indicate that each HBOC has an effective pO2 range in which it unloads the most oxygen, which is dependent on the HBOC's P50. Although it would seem that the HBOC with the lowest P50 (5 mm Hg) should be the best option for O2 delivery to hypoxic tumors, our results indicate that when this is the case, the lowest P50 HBOC yielded insufficient O2 delivery to normoxic tissues (inlet pO2 ∼ 95 mm Hg). Because HBOCs would be transfused into a patient's blood stream before initiating a particular cancer therapy, sufficient oxygen must be delivered to both normoxic and cancerous tissues to maintain normal tissue functions when oxygenating the tumor. Human blood supplemented with a hydrogel‐encapsulated Hb with a P50 of 10 mm Hg was found to deliver sufficient oxygen to normoxic tissue while increasing oxygen delivery to hypoxic regions of tumorgenic tissue. Taken together, this work represents a novel strategy for enhancing the efficacy of existing cancer therapies.
Propagation of embryonic stem cells in stirred suspension without serum
Authors: Kehoe Daniel E. , Lock Lye T. , Parikh Abhirath , Tzanakakis Emmanuel S.
Published: December 2008

Embryonic stem cells (ESCs) with their unlimited capacity for self‐renewal and ability to differentiate along multiple cell lineages are a superb starting material for biotechnology applications and cellular therapies. However, realization of the potential of ESCs requires the development of scalable systems for their production in large quantities and in a regulated manner. Here, we describe a methodology for the expansion of mouse ESCs (mESCs) as pluripotent aggregates in a stirred suspension bioreactor and in medium without serum. Initially, the culture of feeder cell‐independent mESCs in dishes was adapted to serum‐free conditions. Also, we explored whether spinner flasks equipped with a triangle‐shaped impeller and baffles support the culture of mESC aggregates. Serum‐free culture in these vessels resulted in an almost 20‐fold increase in the live mESC concentration over 4 days without significant loss of cell viability. Even after consecutive passages, mESCs retained high expression of pluripotency markers Oct3/4, Rex1 and SSEA‐1. More importantly, when differentiation was induced these cells adopted fates of all three germ layers namely neuroectoderm, cardiac mesoderm and definitive endoderm. These findings demonstrate that stem cells can be propagated under serum‐free conditions in a scalable stirred‐suspension culture without loss of their pluripotency.
In vivo dynamics of glycolysis in Escherichia coli shows need for growth‐rate dependent metabolome analysis
Authors: Schaub Jochen , Reuss Matthias
Published: December 2008

Metabolomics emerges to become an important profiling technique in bio(techno)logical systems. In addition to intracellular metabolite concentrations at (quasi) stationary conditions, stimulus‐response experiments provide information on the dynamic behavior of metabolic pathways. These data are relevant for bioprocess analysis on the level of metabolism and for application of metabolic engineering principles aiming at a metabolic redesign of producer cells. However, even for the well‐studied bacteria Escherichia coli only limited growth‐rate dependent intracellular metabolite information is currently available, thereby impeding comprehensive metabolome analysis. Here, we present intracellular metabolite concentration data of representative glycolytic intermediates in E. coli cultivated in glucose‐limited chemostats, (i) at systematic variation of growth‐rate (D = 0.1, 0.2, 0.3, and 0.4 h−1) and (ii) at both steady‐state and after a glucose pulse applying a recently introduced integrated sampling procedure and LC‐MS analytical method. Whereas intracellular steady‐state concentrations of upper part glycolytic intermediates FBP and DHAP+GAP increased 2.3‐fold, respectively 2.8‐fold, when specific growth‐rate is raised from μ = 0.1 h−1 to μ = 0.4 h−1, the opposite trend was observed for 2PG+3PG and PEP pools with a decrease by a factor of 2.1, respectively 1.9. In glucose pulse experiments FBP and DHAP+GAP showed a 3.3 (1.8)‐fold, respectively 2.8 (2.0)‐fold, increase relative to the steady‐state level at μ = 0.1 (0.4) h−1. Also, the dynamics changed with growth‐rate for these two metabolite pools. In contrast, 2PG+3PG and PEP were characterized by decreased concentrations in response to a glucose pulse and the relative changes related to steady‐state values were significantly smaller compared with FBP and DHAP+GAP. The observed growth‐rate dependency of our data clearly indicates the necessity for metabolome studies covering a broader range of physiological growth conditions.
Screening of a human antibody phage display library against a peptide antigen using stimuli‐responsive bioconjugates
Authors: Stocker Gisela , Dumoulin Dorothée , Vandevyver Caroline , Hilbrig Frank , Freitag Ruth
Published: December 2008

A synthetic antibody phage library (ETH‐2) was screened against the MUC‐1 peptide. Two standard methods were used, namely screening on immunotubes coated with the peptide antigen and on streptavidin‐coated paramagnetic particles together with a biotinylated MUC‐1 peptide. The results were compared with those obtained using a novel approach based on a stimuli‐responsive bioconjugate of avidin and poly‐(N‐isopropylacrylamide), also in combination with the biotinylated peptide. The poly‐(N‐isopropylacrylamide)‐based bioconjugate is soluble below a temperature of 30°C in the aqueous solutions pertinent to this study, but precipitates rapidly once this temperature is surpassed. The process is reversible, and the bioconjugate redissolves once the temperature is lowered again. The stimuli‐responsive bioconjugate was used to isolate specific phage from the library in several rounds of panning, consisting of repeated thermoprecipitation/redissolution cycles in a procedure known as “affinity precipitation.” As far as we know, this is the first time a compound as big as a viral particle has been specifically captured by a stimuli‐responsive bioconjugate. Compared to the two other investigated screening techniques, affinity precipitation of the phage led to a greater variety of genetically different specific phage, while the process was easy to handle and required no dedicated equipment safe for a centrifuge and a thermostated water bath.
Estimation of transgene copy number in transformed citrus plants by quantitative multiplex real‐time PCR
Authors: Omar Ahmad A. , Dekkers Marty G. H. , Graham James H. , Grosser Jude W.
Published: December 2008

Quantitative real‐time PCR (qRT‐PCR) was adapted to estimate transgene copy number of the rice Xa21 gene in transgenic citrus plants. This system used TaqMan qRT‐PCR and the endogenous citrus gene encoding for lipid transfer protein (LTP). Transgenic “Hamlin” sweet orange plants were generated using two different protoplast‐GFP transformation systems: cotransformation and single plasmid transformation. A dilution series of genomic DNA from one of the transgenic lines was used to generate a standard curve for the endogenous LTP and the transgene Xa21. This standard curve was used for relative quantification of the endogenous gene and the transgene. Copy numbers of the transgene Xa21 detected from qRT‐PCR analysis correlated with that from Southern blot analysis (r = 0.834). Thus, qRT‐PCR is an efficient means of estimating copy number in transgenic citrus plants. This analysis can be performed at much earlier stages of transgenic plant development than southern blot analysis, which expedites investigation of transgenes in slow‐growing woody plants.
Automated microfluidic assay system for autoantibodies found in autoimmune diseases using a photoimmobilized autoantigen microarray
Authors: Matsudaira Takahiro , Tsuzuki Saki , Wada Akira , Suwa Akira , Kohsaka Hitoshi , Tomida Maiko , Ito Yoshihiro
Published: December 2008

Autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, and autoimmune diabetes are characterized by the production of autoantibodies that serve as useful diagnostic markers, surrogate markers, and prognostic factors. We devised an in vitro system to detect these clinically pivotal autoantibodies using a photoimmobilized autoantigen microarray. Photoimmobilization was useful for preparing the autoantigen microarray, where autoantigens are covalently immobilized on a plate, because it does not require specific functional groups of the autoantigens and any organic material can be immobilized by a radical reaction induced by photoirradiation. Here, we prepared the microarray using a very convenient method. Aqueous solutions of each autoantigen were mixed with a polymer of poly(ethylene glycol) methacrylate and a photoreactive crosslinker, and the mixtures were microspotted on a plate and dried in air. Finally, the plate was irradiated with an ultraviolet lamp to obtain immobilization. In the assay, patient serum was added to the microarray plate. Antigen‐specific IgG adsorbed on the microspotted autoantigen was detected by peroxidase‐conjugated anti‐IgG antibody. The chemical luminescence intensities of the substrate decomposed by the peroxidase were detected with a sensitive CCD camera. All autoantigens were immobilized stably by this method and used to screen antigen‐specific IgG. In addition, the plate was covered with a polydimethylsiloxane sheet containing microchannels and automated measurement was carried out.
Development and application of an excitation ratiometric optical pH sensor for bioprocess monitoring
Authors: Badugu Ramachandram , Kostov Yordan , Rao Govind , Tolosa Leah
Published: December 2008

The development of a fluorescent excitation ratiometric pH sensor (AHQ‐PEG) using a novel allylhydroxyquinolinium (AHQ) derivative copolymerized with polyethylene glycol dimethacrylate (PEG) is described. The AHQ‐PEG sensor film is shown to be suitable for real‐time, noninvasive, continuous, online pH monitoring of bioprocesses. Optical ratiometric measurements are generally more reliable, robust, inexpensive, and insensitive to experimental errors such as fluctuations in the source intensity and fluorophore photobleaching. The sensor AHQ‐PEG in deionized water was shown to exhibit two excitation maxima at 375 and 425 nm with a single emission peak at 520 nm. Excitation spectra of AHQ‐PEG show a decrease in emission at the 360 nm excitation and an increase at the 420 nm excitation with increasing pH. Accordingly, the ratio of emission at 420:360 nm excitation showed a maximum change between pH 5 and 8 with an apparent pKa of 6.40. The low pKa value is suitable for monitoring the fermentation of most industrially important microorganisms. Additionally, the AHQ‐PEG sensor was shown to have minimal sensitivity to ionic strength and temperature. Because AHQ is covalently attached to PEG, the film shows no probe leaching and is sterilizable by steam and alcohol. It shows rapid (∼2 min) and reversible response to pH over many cycles without any photobleaching. Subsequently, the AHQ‐PEG sensor film was tested for its suitability in monitoring the pH of S. cereviseae (yeast) fermentation. The observed pH using AHQ‐PEG film is in agreement with a conventional glass pH electrode. However, unlike the glass electrode, the present sensor is easily adaptable to noninvasive monitoring of sterilized, closed bioprocess environments without the awkward wire connections that electrodes require. In addition, the AHQ‐PEG sensor is easily miniaturized to fit in microwell plates and microbioreactors for high‐throughput cell culture applications.
Bioprocess optimization using design‐of‐experiments methodology
Authors: Mandenius Carl‐Fredrik , Brundin Anders
Published: December 2008

This review surveys recent applications of design‐of‐experiments (DoE) methodology in the development of biotechnological processes. Methods such as factorial design, response surface methodology, and (DoE) provide powerful and efficient ways to optimize cultivations and other unit operations and procedures using a reduced number of experiments. The multitude of interdependent parameters involved within a unit operation or between units in a bioprocess sequence may be substantially refined and improved by the use of such methods. Other bioprocess‐related applications include strain screening evaluation and cultivation media balancing. In view of the emerging regulatory demands on pharmaceutical manufacturing processes, exemplified by the process analytical technology (PAT) initiative of the United States Food and Drug Administration, the use of experimental design approaches to improve process development for safer and more reproducible production is becoming increasingly important. Here, these options are highlighted and discussed with a few selected examples from antibiotic fermentation, expanded bed optimization, virus vector transfection of insect cell cultivation, feed profile adaptation, embryonic stem cell expansion protocols, and mammalian cell harvesting.
Assessment of potential life‐cycle energy and greenhouse gas emission effects from using corn‐based butanol as a transportation fuel
Authors: Wu May , Wang Michael , Liu Jiahong , Huo Hong
Published: December 2008

Since advances in the ABE (acetone‐butanol‐ethanol) fermentation process in recent years have led to significant increases in its productivity and yields, the production of butanol and its use in motor vehicles have become an option worth evaluating. This study estimates the potential life‐cycle energy and emission effects associated with using bio‐butanol as a transportation fuel. It employs a well‐to‐wheels (WTW) analysis tool: the Greenhouse Gases, Regulated Emissions, and Energy Use in Transportation (GREET) model. The estimates of life‐cycle energy use and greenhouse gas (GHG) emissions are based on an Aspen Plus® simulation for a corn‐to‐butanol production process, which describes grain processing, fermentation, and product separation. Bio‐butanol‐related WTW activities include corn farming, corn transportation, butanol production, butanol transportation, and vehicle operation. In this study, we also analyzed the bio‐acetone that is coproduced with bio‐butanol as an alternative to petroleum‐based acetone. We then compared the results for bio‐butanol with those of conventional gasoline. Our study shows that driving vehicles fueled with corn‐based butanol produced by the current ABE fermentation process could result in substantial fossil energy savings (39%–56%) and avoid large percentage of the GHG emission burden, yielding a 32%–48% reduction relative to using conventional gasoline. On energy basis, a bushel of corn produces less liquid fuel from the ABE process than that from the corn ethanol dry mill process. The coproduction of a significant portion of acetone from the current ABE fermentation presents a challenge. A market analysis of acetone, as well as research and development on robust alternative technologies and processes that minimize acetone while increase the butanol yield, should be conducted.
A hybrid model of anaerobic E. coli GJT001: Combination of elementary flux modes and cybernetic variables
Authors: Kim Jin Il , Varner Jeffery D. , Ramkrishna Doraiswami
Published: December 2008

Flux balance analysis (FBA) in combination with the decomposition of metabolic networks into elementary modes has provided a route to modeling cellular metabolism. It is dependent, however, on the availability of external fluxes such as substrate uptake or growth rate before estimates can become available of intracellular fluxes. The framework classically does not allow modeling of metabolic regulation or the formulation of dynamic models except through dynamic measurement of external fluxes. The cybernetic modeling approach of Ramkrishna and coworkers provides a dynamic framework for modeling metabolic systems because of its focus on describing regulatory processes based on cybernetic arguments and hence has the capacity to describe both external and internal fluxes. In this article, we explore the alternative of developing hybrid models combining cybernetic models for the external fluxes with the flux balance approach for estimation of the internal fluxes. The approach has the merit of the simplicity of the early cybernetic models and hence computationally facile while also providing detailed information on intracellular fluxes. The hybrid model of this article is based on elementary mode decomposition of the metabolic network. The uptake rates for the various elementary modes are combined using global cybernetic variables based on maximizing substrate uptake rates. Estimation of intracellular metabolism is based on its stoichiometric coupling with the external fluxes under the assumption of (pseudo‐) steady state conditions. The set of parameters of the hybrid model was estimated with the aid of nonlinear optimization routine, by fitting simulations with dynamic experimental data on concentrations of biomass, substrate, and fermentation products. The hybrid model estimations were tested with FBA (based on measured substrate uptake rate) for two different metabolic networks (one is a reduced network which fixes ATP contribution to the biomass and maintenance requirement of ATP, and the other network is a more complex network which has a separate reaction for maintenance.) for the same experiment involving anaerobic growth of E. coli GJT001. The hybrid model estimated glucose consumption and all fermentation byproducts to better than 10%. The FBA makes similar estimations of fermentation products, however, with the exception of succinate. The simulation results show that the global cybernetic variables alone can regulate the metabolic reactions obtaining a very satisfactory fit to the measured fermentation byproducts. In view of the hybrid model's ability to predict biomass growth and fermentation byproducts of anaerobic E. coli GJT001, this reduced order model offers a computationally efficient alternative to more detailed models of metabolism and hence useful for the simulation of bioreactors.
Liquefaction, saccharification, and fermentation of ammoniated corn to ethanol
Authors: Taylor Frank , Kim Tae Hyun , Abbas Charles A. , Hicks Kevin B.
Published: December 2008

Treatment of whole corn kernels with anhydrous ammonia gas has been proposed as a way to facilitate the separation of nonfermentable coproducts before fermentation of the starch to ethanol, but the fermentability of ammoniated corn has not been thoroughly investigated. Also, it is intended that the added ammonia nitrogen in ammonia treated corn (∼1 g per kg corn) may satisfy the yeast nutritional requirement for free amino nitrogen (FAN). In this study, procedures for ammoniation, liquefaction, saccharification, and fermentation at two scales (12‐L and 50‐mL) were used to determine the fermentation rate, final ethanol concentration, and ethanol yield from starch in ammoniated or nonammoniated corn. The maximum achievable ethanol concentration at 50 h fermentation time was lower with ammoniated corn than with nonammoniated corn. The extra nitrogen in ammoniated corn satisfied some of the yeast requirements for FAN, thereby reducing the requirement for corn steep liquor. Based upon these results, ammoniation of corn does not appear to have a positive impact on the fermentability of corn to ethanol. Ammoniation may still be cost effective, if the advantages in terms of improved separations outweigh the disadvantages in terms of decreased fermentability.
Different effects of L‐arginine on protein refolding: Suppressing aggregates of hydrophobic interaction, not covalent binding
Authors: Chen Jing , Liu Yongdong , Wang Yinjue , Ding Hong , Su Zhiguo
Published: December 2008

Arginine is one of the most favorable additives in protein refolding. However, arginine does not work for certain disulfide‐bond‐containing proteins, which is not yet well explained. In this work, refolding of three proteins in the presence of 0–2 M arginine was investigated and compared. Bovine carbonic anhydrase B (CAB), containing no cysteine, was successfully refolded with the help of arginine. The refolding yield could reach almost 100% in the presence of 0.75 M arginine. However, recombinant human colony stimulating factor (rhG‐CSF), containing five cysteines, could only achieve 65% refolding yield. The formation of aggregates was found. Blocking of free SH groups of the denatured rhG‐CSF by iodoacetamide and subsequently refolding of the protein could reduce the aggregate formation substantially. Further investigation on recombinant green fluorescence protein (GFP), containing two cysteines, also revealed the accumulation of oligomers. The content of oligomers increased with the concentration of arginine, reaching about 30% at 2 M arginine. Comparison of reduced and nonreduced SDS‐PAGE revealed that the oligomers were formed through intermolecular disulfide binding. Analysis of the refolding kinetics indicated that intermolecular disulfide bonds were probably formed in the intermediate stage where arginine slowed down the refolding rate and stabilized the intermediates. The accumulated intermediates with unpaired cysteine possessed more chances to react with each other to form oligomers, whereas arginine failed to inhibit disulfide bond formation.
O‐GLcNAc post‐translational modifications regulate the entry of neurons into an axon branching program
Authors: Francisco Herb , Kollins Katherine , Varghis Neal , Vocadlo David , Vosseller Keith , Gallo Gianluca
Published: December 2008

Many neuronal cytosolic and nuclear proteins are post‐translationally modified by the reversible addition of O‐linked N‐acetylglucosamine (O‐GlcNAc) on serines and threonines. The cellular functions of O‐GlcNAc modifications in neuronal development are not known. We report that O‐GlcNAc‐modified proteins are distributed nonuniformly throughout cultured primary chicken forebrain neurons, with intense immunostaining of the cell body, punctuate immunostaining in axons and all processes, and localization in filopodia/lamellipodia. Overexpression of O‐GlcNAcase, the enzyme that removes O‐GlcNAc from proteins, increased the percentage of neurons exhibiting axon branching without altering the frequency of axon branches on a per neuron basis and increased the numbers of axonal filopodia. Conversely, pharmacologically increasing O‐GlcNAc levels on proteins through specific inhibition of O‐GlcNAcase with the inhibitor 9d decreased the numbers of axonal filopodia, but had no effect on axon length or branching. Treatment with an alternative O‐GlcNAcase inhibitor, PUGNAc, similarly decreased the number of axonal filopodia. Furthermore, axon branching induced by the adenylyl cyclase activator forskolin was suppressed by pharmacological inhibition of O‐GlcNAcase. Western analysis revealed that O‐GlcNAc levels regulate the phosphorylation of some PKA substrates in response to forskolin. These data provide the first evidence of O‐GlcNAc modification‐specific influences in neuronal development in primary culture, and indicate specific roles for O‐GlcNAc in the regulation of axon morphology. © 2008 Wiley Periodicals, Inc. Develop Neurobiol, 2009