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A central concern of biopharmaceutical R&D is the production of sufficient quantities of recombinant products from manufacturing processes based on animal cell culture. The way in which bioprocess researchers have addressed this question experienced a tremendous shift over the years, progressing from almost empirical to more rational approaches. A step further is the application of systems biotechnology: recent technological advances for large-scale cell state characterization and creative methods for host cell modeling are becoming crucial for next-generation bioprocess optimization. Here we provide an overview of the main trends towards this goal, with a focus on metabolic models as central scaffolds for data integration and prediction of bioprocess outcomes. 相似文献
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Rodrigues ML Archer M Martel P Miranda S Thomaz M Enguita FJ Baptista RP Pinho e Melo E Sousa N Cravador A Carrondo MA 《Biochimica et biophysica acta》2006,1764(1):110-121
The crystal structure of the elicitin beta-cinnamomin (beta-CIN) was determined in complex with ergosterol at 1.1 A resolution. beta-CIN/ergosterol complex crystallized in the monoclinic space group P2(1), with unit cell parameters of a = 31.0, b = 62.8, c = 50.0 A and beta = 93.4 degrees and two molecules in the asymmetric unit. Ligand extraction with chloroform followed by crystallographic analysis yielded a 1.35 A structure of beta-CIN (P4(3)2(1)2 space group) where the characteristic elicitin fold was kept. After incubation with cholesterol, a new complex structure was obtained, showing that the protein retains, after the extraction procedure, its ability to complex sterols. The necrotic effect of beta-CIN on tobacco was also shown to remain unchanged. Theoretical docking studies of the triterpene lupeol to beta-CIN provided an explanation for the apparent inability of beta-CIN to bind this ligand, as observed experimentally. 相似文献
66.
1,3-Propanediol Dehydrogenase from Klebsiella pneumoniae: Decameric Quaternary Structure and Possible Subunit Cooperativity 总被引:1,自引:0,他引:1
David Maral Ana Toste Rêgo Maria Armnia Carrondo Francisco J. Enguita 《Journal of bacteriology》2009,191(4):1143-1151
Klebsiella pneumoniae is a nosocomial pathogen frequently isolated from opportunistic infections, especially in clinical environments. In spite of its potential pathogenicity, this microorganism has several metabolic potentials that could be used in biotechnology applications. K. pneumoniae is able to metabolize glycerol as a sole source of carbon and energy. 1,3-Propanediol dehydrogenase is the core of the metabolic pathway for the use of glycerol. We have determined the crystallographic structure of 1,3-propanediol dehydrogenase, a type III Fe-NAD-dependent alcohol dehydrogenase, at 2.7-Å resolution. The structure of the enzyme monomer is closely related to that of other alcohol dehydrogenases. The overall arrangement of the enzyme showed a decameric structure, formed by a pentamer of dimers, which is the catalytic form of the enzyme. Dimers are associated by strong ionic interactions that are responsible for the highly stable in vivo packing of the enzyme. Kinetic properties of the enzyme as determined in the article would suggest that this decameric arrangement is related to the cooperativity between monomers. 相似文献
67.
Carinhas N Bernal V Yokomizo AY Carrondo MJ Oliveira R Alves PM 《Applied microbiology and biotechnology》2009,81(6):1041-1049
One of the major concerns regarding the use of insect cells and baculovirus expression vectors for the production of recombinant
proteins is the drop in production observed when infecting cultures at high cell densities; this work attempts to understand
this so-called cell density effect in the scope of baculovirus production for gene therapy purposes. A Spodoptera frugiperda insect cell line (Sf-9) was cultured and infected in serum-free medium, and the patterns of production of a recombinant baculovirus
expressing the green fluorescent protein (GFP) were analyzed at different cell concentrations at infection (CCIs) and multiplicities
of infection (MOIs). The results confirm that a cell density effect on productivity occurs which is dependent on the MOI used,
with a high MOI “delaying” the drop in production to higher cell densities. Medium replacement at the time of infection using
a high MOI considerably improved baculovirus production, with the different production indicators, namely the titer, specific
yield, amplification factor, and time of harvesting, increasing with cell concentration for the CCI range tested. Virus titers
as high as 2.6 × 1010 IP.mL−1 were obtained in cultures infected at 3.5 × 106 cells.mL−1, while the amplification factor was roughly 19 times higher than the highest value obtained without medium exchange. 相似文献
68.
Advances in on-line monitoring and control of mammalian cell cultures: Supporting the PAT initiative
In recent years, much attention has been directed towards the development of global methods for on-line process monitoring, especially since the Food and Drug Administration (FDA) launched the Process Analytical Technology (PAT) guidance, stimulating biopharmaceutical companies to update their monitoring tools to ensure a pre-defined final product quality. The ideal technologies for biopharmaceutical processes should operate in situ, be non-invasive and generate on-line information about multiple key bioprocess and/or metabolic variables. A wide range of spectroscopic techniques based on in situ probes have already been tested in mammalian cell cultures, such as near infrared (NIR), mid infrared (MIR), 2D fluorescence and dielectric capacitance spectroscopy; similarly, the electronic nose technique based on chemical array sensors has been tested for in situ off-gas analysis of mammalian cell cultures. All these methods provide series of spectra, from which meaningful information must be extracted. In this sense, data mining techniques such as principal components regression (PCR), partial least squares (PLS) or artificial neural networks (ANN) have been applied to handle the dense flow of data generated from the real-time process analyzers. Furthermore, the implementation of feedback control methods would help to improve process performance and ultimately ensure reproducibility. This review discusses the suitability of several spectroscopic techniques coupled with chemometric methods for improved monitoring and control of mammalian cell processes. 相似文献
69.
Maria Candida M. Mellado Jimmy A. Mena António Lopes Octavio T. Ramírez Manuel J.T. Carrondo Laura A. Palomares Paula M. Alves 《Biotechnology and bioengineering》2009,104(4):674-686
Virus‐like particles constitute potentially relevant vaccine candidates. Nevertheless, their behavior in vitro and assembly process needs to be understood in order to improve their yield and quality. In this study we aimed at addressing these issues and for that purpose triple‐ and double‐layered rotavirus‐like particles (TLP 2/6/7 and DLP 2/6, respectively) size and zeta potential were measured using dynamic light scattering at different physicochemical conditions, namely pH, ionic strength, and temperature. Both TLP and DLP were stable within a pH range of 3–7 and at 5–25°C. Aggregation occurred at 35–45°C and their disassembly became evident at 65°C. The isoelectric points of TLP and DLP were 3.0 and 3.8, respectively. In vitro kinetics of TLP disassembly was monitored. Ionic strength, temperature, and the chelating agent employed determined disassembly kinetics. Glycerol (10%) stabilized TLP by preventing its disassembly. Disassembled TLP was able to reassemble by dialysis at high calcium conditions. VP7 monomers were added to DLP in the presence of calcium to follow in vitro TLP assembly kinetics; its assembly rate being mostly affected by pH. Finally, DLP and TLP were found to coexist under certain conditions as determined from all reaction products analyzed by capillary electrophoresis. Overall, these results contribute to the design of new strategies for the improvement of TLP yield and quality by reducing the VP7 detachment from TLP. Biotechnol. Bioeng. 2009; 104: 674–686 © 2009 Wiley Periodicals, Inc. 相似文献
70.
Screening of novel excipients for improving the stability of retroviral and adenoviral vectors 总被引:1,自引:0,他引:1
In the past decade there has been an increase in the application of viral vectors in the laboratory and clinical trials of human gene therapy, retroviral and adenoviral vectors among the most used. However, the limited stability of these vectors creates problems in the design of experiments, transport, and storage. Vectors stored at -80 degrees C must be quickly shipped on dry ice, which is somewhat cumbersome. Alternatively, viral vectors can be preserved in a lyophilized form. However, loss of viral activity during lyophilization can also be a serious problem. In this report we identify novel candidate formulations containing new compatible solutes, ectoin, hydroxyectoin, and firoin, that allow better stability of retroviral and adenoviral vectors during storage. For retroviral vectors, the maximum stabilization for long-term storage was achieved through lyophilization followed by storage at -20 degrees C using a formulation of Tris buffer pH 7.2 containing firoin (0.5 M), a half-life of 340 days being obtained. Adenoviral vectors storage at -80 degrees C in solution using Tris buffer pH 8.0 with firoin was the best method for long-term storage, with a half-life exceeding 1 year. 相似文献