bluuebunny/crossref_metadata_2025_split huggingface dataset:
Spatiotemporal modeling and analysis of transient gene delivery
Authors: Jandt Uwe , Shao Shi , Wirth Manfred , Zeng An‐Ping 
Published: May 2011

AbstractA quantitative and mechanistic understanding of intracellular transport processes in eukaroytic cells during transient transfection is an important prerequisite for the systematic and specific optimization of transient gene expression procedures for pharmaceutic and industrial protein production. There is evidence that intracellular transport processes during gene delivery and their regulation may have significant influence on the transfection efficiency. This contribution describes a compartmented, spatiotemporally resolved and stochastic modeling approach that describes intracellular transport processes responsible for gene delivery during transient transfection. It enables a detailed prediction and analysis and identification of potential bottlenecks. This model is currently being adapted to a model cell line, HEK293s. The simulated results are compared with experimental quantitative polymerase chain reaction (qPCR) data and confocal imaging data obtained with transfected and stained HEK293 cells. Global parameter estimation is performed to qPCR data based on two different novel plasmid constructs in order to identify candidates for plasmid‐specific transport parameter variations. The influence of the specific property of HEK293 cells to grow in clusters is investigated and the impact of active microtubule transport depending on cell morphology and clustering is examined. A general sensitivity analysis allows for the identification of the sensitive parameters. Biotechnol. Bioeng. 2011;108:2205–2217. © 2011 Wiley Periodicals, Inc.
A mechanistic understanding of production instability in CHO cell lines expressing recombinant monoclonal antibodies
Authors: Kim Minsoo , O'Callaghan Peter M. , Droms Kurt A. , James David C. 
Published: May 2011

AbstractOne of the most significant problems in industrial bioprocessing of recombinant proteins using engineered mammalian cells is the phenomenon of cell line instability, where a production cell line suffers a loss of specific productivity (qP). This phenomenon occurs with unpredictable kinetics and has been widely observed in Chinese hamster ovary (CHO) cell lines and with all commonly used gene expression systems. The underlying causes (both genetic and physiological) and the precise molecular mechanisms underpinning cell line instability have yet to be fully elucidated, although recombinant gene silencing and loss of recombinant gene copies have been shown to cause qP loss. In this work we have investigated the molecular mechanisms underpinning qP instability over long‐term sub‐culture in CHO cell lines producing recombinant IgG1 and IgG2 monoclonal antibodies (Mab's). We demonstrate that production instability derives from two primary mechanisms: (i) epigenetic—methylation‐induced transcriptional silencing of the CMV promoter driving Mab gene transcription and (ii) genetic—progressive loss of recombinant Mab gene copies in a proliferating CHO cell population. We suggest that qP decline resulting from loss of recombinant genes is a consequence of the inherent genetic instability of recombinant CHO cell lines. Biotechnol. Bioeng. 2011;108: 2434–2446. © 2011 Wiley Periodicals, Inc.
Microprinted feeder cells guide embryonic stem cell fate
Authors: Tavana Hossein , Mosadegh Bobak , Zamankhan Parsa , Grotberg James B. , Takayama Shuichi 
Published: May 2011

AbstractWe introduce a non‐contact approach to microprint multiple types of feeder cells in a microarray format using immiscible aqueous solutions of two biopolymers. Droplets of cell suspension in the denser aqueous phase are printed on a substrate residing within a bath of the immersion aqueous phase. Due to their affinity to the denser phase, cells remain localized within the drops and adhere to regions of the substrate underneath the drops. We show the utility of this technology for creating duplex heterocellular stem cell niches by printing two different support cell types on a gel surface and overlaying them with mouse embryonic stem cells (mESCs). As desired, the type of printed support cell spatially direct the fate of overlaid mESCs. Interestingly, we found that interspaced mESCs colonies on differentiation‐inducing feeder cells show enhanced neuronal differentiation and give rise to dense networks of neurons. This cell printing technology provides unprecedented capabilities to efficiently identify the role of various feeder cells in guiding the fate of stem cells. Biotechnol. Bioeng. 2011;108: 2509–2516. © 2011 Wiley Periodicals, Inc.
Clonal analysis of the proliferation potential of human bone marrow mesenchymal stem cells as a function of potency
Authors: Russell Katie C. , Lacey Michelle R. , Gilliam Jennifer K. , Tucker H. Alan , Phinney Donald G. , O'Connor Kim C. 
Published: May 2011

AbstractHuman mesenchymal stem cells (MSCs) from bone marrow are a heterogeneous ensemble of progenitors and lineage‐committed cells, with a broad range of regenerative properties. Ex vivo expansion to produce sufficient quantities of MSCs is essential for most therapeutic applications. The present study resolves the relationship between proliferation potential of MSCs and their potency. Clonal analysis generated single‐cell derived colonies of MSCs that were classified according to their trilineage potential to exhibit adipo‐ (A), chondro‐ (C), and osteogenesis (O) as a measure of potency. Multipotent OAC clones were highly proliferative with colony‐forming efficiencies that ranged from 35% to 90%; whereas, O clones formed colonies with an efficiency of 5% or less (P < 0.01). Similar trends were evident during ex vivo expansion: for example, the median specific growth rate was 0.85 day−1 (20 h doubling time) for cultures inoculated with OAC clones and was 5‐fold less for inocula of O clones (P < 0.01). OA and OC clones had similar proliferation potentials. More than 75% of cells in subconfluent cultures inoculated with O clones stained positive for senescence‐associated β‐galactosidase activity vs. less than 10% for OAC clones (P < 0.001). Apoptotic cells were in the minority for all potency groups. Preliminary data generated during clonal analysis suggest that osteogenic potential of MSCs to produce mineralized matrix is a function of potency, as well. These results are discussed in the context of the preparation of efficacious MSC therapies by ex vivo expansion. Biotechnol. Bioeng. 2011;108: 2716–2726. © 2011 Wiley Periodicals, Inc.
Effect of O2:CO2 ratio on the primary metabolism of Chlamydomonas reinhardtii
Authors: Kliphuis Anna M.J. , Martens Dirk E. , Janssen Marcel , Wijffels René H. 
Published: May 2011

AbstractHigh oxygen:carbon dioxide ratios may have a negative effect on growth and productivity of microalgae. To investigate the effect of O2 and CO2 concentrations and the ratio between these on the metabolism of Chlamydomonas reinhardtii we performed turbidostat experiments at different O2:CO2 ratios. These experiments showed that elevated O2 concentrations and the corresponding increase in the ratio of O2:CO2 common in photobioreactors led to a reduction of growth and biomass yield on light with 20–30%. This is most probably related to the oxygenase activity of Rubisco and the resulting process of photorespiration. Using metabolic flux modeling with measured rates for each experiment we were able to quantify the ratio of the oxygenase reaction to the carboxylase reaction of Rubisco and could demonstrate that photorespiration indeed can cause the reduction in biomass yield on light. The calculated ratio of the oxygenase reaction to the carboxylase reaction was 16.6% and 20.5% for air with 2% CO2 and 1% CO2, respectively. Thus photorespiration has a significant impact on the biomass yield on light already at conditions common in photobioreactors (air with 2% CO2). Biotechnol. Bioeng. 2011;108: 2390–2402. © 2011 Wiley Periodicals, Inc.
Predicting mab product yields from cultivation media components, using near‐infrared and 2D‐fluorescence spectroscopies
Authors: Jose Gledson E. , Folque Francisca , Menezes Jose C. , Werz Silke , Strauss Ulrike , Hakemeyer Christian 
Published: May 2011

AbstractThe yield of monoclonal antibody (Mab) production processes depends on media formulation, inocula quality, and process conditions. As in industrial processes tight cultivation conditions are used, and inocula quality and viable cell densities are controlled to reasonable levels, media formulation and raw materials lot‐to‐lot variability in quality will have, in those circumstances, the highest impact on process performance. In the particular Mab process studied, two different raw materials were used: a complex carbon and nitrogen source made of specific peptones and defined chemical media containing multiple components. Using different spectroscopy techniques for each of the raw material types, it was concluded that for the complex peptone‐based ingredient, near‐infrared (NIR) spectroscopy was more capable of capturing lot‐to‐lot variability. For the chemically defined media containing fluorophores, two‐dimensional (2D)‐fluorescence spectroscopy was more capable of capturing lot‐to‐lot variability. Because in Mab cultivation processes both types of raw materials are used, combining the NIR and 2D‐fluorescence spectra for each of the media components enabled predictive models for yield to be developed that out‐performed any other model involving either one raw material alone, or only one type of spectroscopic tool for both raw materials. For each particular raw material, the capability of each spectroscopy to detect lot‐to‐lot differences was demonstrated after spectra preprocessing and specific wavelength regions selection. The work described and the findings reported here open up several possibilities that could be used to feed‐forward control the process. These include, for example, enabling specific actions to be taken regarding media formulation with particular lots, and all types of predictive control actions aimed at increasing batch‐to‐batch yield and product quality consistency at harvest. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Process and economic evaluation for monoclonal antibody purification using a membrane‐only process
Authors: Varadaraju Hemanthram , Schneiderman Steven , Zhang Lifeng , Fong Hao , Menkhaus Todd J. 
Published: May 2011

AbstractIn recent years, the market for therapeutic monoclonal antibodies (mAb) has grown exponentially, and with this there has been a desire to reduce the costs associated with production and purification of these high‐value biological products. A typical mAb purification process involves three adsorption/chromatography steps [protein A, ion exchange (IEX), and hydrophobic interaction (HIC)], along with ultrafiltration, nanofiltration, and microfiltration. With the development of membrane adsorption/chromatography as a viable alternative to traditional pack bed systems, the opportunity exists to complete the entire downstream purification process using only membrane operations. In this study, the process simulation tool SuperPro Designer was used to evaluate the application of recently developed ultra‐high capacity electrospun nanofibrous adsorption membranes as a replacement for conventional chromatographic media in the downstream mAb production process. The simulation showed that nanofibrous adsorption membranes in place of the three packed bed chromatography steps reduced the required volume of protein A, IEX, and HIC adsorptive medium by 25, 80, and 80%, respectively. In addition, the membrane‐only process reduced the downstream processing time by 50%, decreased the number of labor hours associated with the purification steps by 40%, generated 40% less aqueous waste, and reduced the overall downstream process operating expenses per unit product by 23%. There were also significant savings in facility construction costs and the price of fixed equipment required for separations. With these savings not only is the membrane‐only process economically competitive with the traditional packed bed operations, but it offers the possibility of moving toward more disposable process.© 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Assembly of horseradish peroxidase within supported cationic bilayers
Authors: Leão‐Silva Ana Carolina , Naves Alliny F. , Pereira Edla M. A. , Petri Denise F. S. , Carmona‐Ribeiro Ana M. 
Published: May 2011

AbstractThe interaction between horseradish peroxidase (HRP) and dioctadecyldimethylammonium bromide (DODAB) bilayers supported on polystyrene microspheres (PSS) or on flat silicon wafers was evaluated from the following techniques: (1) dynamic light‐scattering for determining size distributions, zeta‐potentials and polydispersities for dispersions; (2) spectrophotometric determination of HRP concentration in supernatants of centrifuged mixtures; (3) in situ ellipsometry for mean thickness of deposited layers on wafers; (4) kinetics of product appearance for oxidation of 2,2′‐azino‐bis‐3‐ethylbenzothiazoline‐6‐sulfonic acid by H2O2 in presence of free or immobilized enzyme. HRP incorporation (3.0 mg/m2) did not alter mean diameter and zeta‐potential of PSS/DODAB particles but reduced enzyme activity by 50%, though activity persisted after several rinsing steps. In situ ellipsometry could not detect any HRP layer on top of the DODAB bilayer. HRP insertion in the bilayer core explained all results for both systems. Useful biotechnological applications are anticipated for such assemblies. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
High‐pressure refolding of human vascular endothelial growth factor (VEGF) recombinantly expressed in bacterial inclusion bodies: Refolding optimization, and feasibility assessment
Authors: Cothran Amber , John Richard J. St. , Schmelzer Charles H. , Pizarro Shelly A. 
Published: May 2011

AbstractHigh‐pressure has been established as an effective technique for refolding proteins at high concentrations. In this study, high hydrostatic pressure (1–3 kbar) was utilized to refold a homodimeric protein from inclusion bodies and the process was evaluated for large‐scale manufacturing feasibility. This research focused on increasing protein concentration while maximizing yield and product quality. Refolding yields of 29–42% were achieved in the absence of urea at 2 kbar and at a protein concentration of 6 g/L. Optimization of the refolding buffer composition via multivariate design of experiments and other process parameters such as refolding pressure, gas sparging, and time under pressure are discussed. Although high‐pressure refolding can be considered a viable technology for manufacturing if the gains are clearly identified, in this particular case, the benefits that the high‐pressure technology offers do not compensate for the drawbacks of implementing new equipment in an existing facility, and unknown impact of scale‐up for this molecule. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Protein adsorption and transport in cation exchangers with a rigid backbone matrix with and without polymeric surface extenders
Authors: Almodóvar Ernie X. Pérez , Tao Yinying , Carta Giorgio 
Published: May 2011

AbstractWe compare the properties and protein adsorption characteristics of two polymeric cation exchangers: UNOsphere S, which has an open macroporous architecture, and Nuvia S, which is based on a very similar backbone matrix but contains sulfonated polymeric surface extenders. A monoclonal IgG and lysozyme were used as model adsorbates. The characteristic pore sizes, determined by inverse size exclusion chromatography, were about 140 nm for UNOsphere S, and only about 10 nm for Nuvia S, indicating that the polymeric extenders occupy a substantial portion of the base matrix pores. Greater exclusion limits were found for Nuvia S in 1 M NaCl and for a similar matrix containing uncharged surface extenders, suggesting that the polymeric extenders collapse partially at high ionic strength or when they are uncharged. Large equilibrium binding capacities were obtained for Nuvia S, approaching 320 ± 10 mg/mL of particle volume for both proteins in comparison with the UNOsphere S values of 170 ± 10 and 120 ± 10 mg/mL for lysozyme and IgG, respectively. Much higher adsorption rates were also found for Nuvia S, and the rate was nearly independent of protein concentration in solution. Confocal laser scanning microscopy showed very sharp intraparticle protein concentration profiles for UNOsphere S, consistent with a pore diffusion mechanism but diffuse concentration profiles for Nuvia S, consistent with a solid diffusion mechanism. The improved capacity and transport afforded by the polymeric extenders provide substantial potential benefits for bioprocess applications without sacrificing the desirable flow properties of the backbone matrix. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Assessment of the manufacturability of Escherichia coli high cell density fermentations
Authors: Perez‐Pardo M. A. , Ali S. , Balasundaram B. , Mannall G. J. , Baganz F. , Bracewell D. G. 
Published: May 2011

AbstractThe physical and biological conditions of the host cell obtained at the end of fermentation influences subsequent downstream processing unit operations. The ability to monitor these characteristics is central to the improvement of biopharmaceutical manufacture. In this study, we have used a combination of techniques such as adaptive focus acoustics (AFA) and ultra scale‐down (USD) centrifugation that utilize milliliter quantities of sample to obtain an insight into the interaction between cells from the upstream process and initial downstream unit operations. This is achieved primarily through an assessment of cell strength and its impact on large‐scale disc stack centrifugation performance, measuring critical attributes such as viscosity and particle size distribution. An Escherichia coli fed‐batch fermentation expressing antibody fragments in the periplasm was used as a model system representative of current manufacturing challenges. The weakening of cell strength during cultivation time, detected through increased micronization and viscosity, resulted in a 2.6‐fold increase in product release rates from the cell (as measured by AFA) and approximately fourfold decrease in clarification performance (as measured by USD centrifugation). The information obtained allows for informed harvest point decisions accounting for both product leakages during fermentation and potential losses during primary recovery. The clarification performance results were verified at pilot scale. The use of these technologies forms a route to the process understanding needed to tailor the host cell and upstream process to the product and downstream process, critical to the implementation of quality‐by‐design principles. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Estimating cell specific oxygen uptake and carbon dioxide production rates for mammalian cells in perfusion culture
Authors: Goudar Chetan T. , Piret James M. , Konstantinov Konstantinov B. 
Published: May 2011

AbstractWe present robust methods for online estimation of cell specific oxygen uptake and carbon dioxide production rates (qO2 and qCO2, respectively) during perfusion cultivation of mammalian cells. Perfusion system gas and liquid phase mass balance expressions for oxygen and carbon dioxide were used to estimate qO2, qCO2 and the respiratory quotient (RQ) for Chinese hamster ovary (CHO) cells in perfusion culture over 12 steady states with varying dissolved oxygen (DO), pH, and temperature set points. Under standard conditions (DO = 50%, pH = 6.8, T = 36.5°C), qO2 and qCO2 ranges were 5.14–5.77 and 5.31–6.36 pmol/cell day, respectively, resulting in RQ values of 0.98–1.14. Changes to DO had a slight reducing effect on respiration rates with qO2 and qCO2 values of 4.64 and 5.47, respectively, at DO = 20% and 4.57 and 5.12 at DO = 100%. Respiration rates were lower at low pH with qO2 and qCO2 values of 4.07 and 4.15 pmol/cell day at pH = 6.6 and 4.98 and 5.36 pmol/cell day at pH = 7. Temperature also impacted respiration rates with respective qO2 and qCO2 values of 3.97 and 4.02 pmol/cell day at 30.5°C and 5.53 and 6.25 pmol/cell day at 37.5°C. Despite these changes in qO2 and qCO2 values, the RQ values in this study ranged from 0.98 to 1.23 suggesting that RQ was close to unity. Real‐time qO2 and qCO2 estimates obtained using the approach presented in this study provide additional quantitative information on cell physiology both during bioprocess development and commercial biotherapeutic manufacturing. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Development of a mathematical model for evaluating the dynamics of normal and apoptotic Chinese hamster ovary cells
Authors: Naderi Saeideh , Meshram Mukesh , Wei Catherine , McConkey Brendan , Ingalls Brian , Budman Hector , Scharer Jeno 
Published: May 2011

AbstractA metabolic flux based methodology was developed for modeling the metabolism of a Chinese hamster ovary cell line. The elimination of insignificant fluxes resulted in a simplified metabolic network which was the basis for modeling the significant metabolites. Employing kinetic rate expressions for growing and non‐growing subpopulations, a logistic model was developed for cell growth and dynamic models were formulated to describe culture composition and monoclonal antibody (MAb) secretion. The model was validated for a range of nutrient concentrations. Good agreement was obtained between model predictions and experimental data. The ultimate goal of this study is to establish a comprehensive dynamic model which may be used for model‐based optimization of the cell culture for MAb production in both batch and fed‐batch systems. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011
Synthesis and performance of 3D‐Megaporous structures for enzyme immobilization and protein capture
Authors: Bibi Noor Shad , Gavara Poondi Rajesh , Espinosa Silvia L. Soto , Grasselli Mariano , Fernández‐Lahore Marcelo 
Published: May 2011

AbstractThe preparation of megaporous bodies, with potential applications in biotechnology, was attempted by following several strategies. As a first step, naive and robust scaffolds were produced by polymerization of selected monomers in the presence of a highly soluble cross‐linker agent. Ion‐exchange function was incorporated by particle embedding, direct chemical synthesis, or radiation‐induced grafting. The total ionic capacity of such systems was 1.5 mmol H+/g, 1.4 mmol H+/g, and 17 mmol H+/g, respectively. These values were in agreement with the ability to bind model proteins: observed dynamic binding capacity at 50% breakthrough was ≅7.2 mg bovine serum albumin/g, ≅7.4 hen egg‐white lysozyme (HEWL) mg/g, and ≅108 HEWL mg/g. In the later case, total (static) binding capacity reached 220 mg/g. It was observed that the structure and size of the megapores remained unaffected by the grafting procedure which, however, allowed for the highest protein binding capacity. Lysozyme supported on grafted body showed extensive clarification activity against a Micrococcus lysodekticus suspension in the flow‐through mode, i.e., 90% destruction of suspended microbial cells was obtained with a residence time ≈ 18 min. Both protein capture and biocatalysis applications are conceivable with the 3D‐megaporous materials described in this work. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011