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21.
Aponte-Ubillus Juan Jose Barajas Daniel Peltier Joseph Bardliving Cameron Shamlou Parviz Gold Daniel 《Applied microbiology and biotechnology》2018,102(3):1045-1054
Applied Microbiology and Biotechnology - Recombinant adeno-associated virus (rAAV) vectors are increasingly popular tools for gene therapy applications. Their non-pathogenic status, low... 相似文献
22.
Miesegaes GR Lute S Strauss DM Read EK Venkiteshwaran A Kreuzman A Shah R Shamlou P Chen D Brorson K 《Biotechnology and bioengineering》2012,109(8):2048-2058
Traditionally, post-production culture harvest capture of therapeutic monoclonal antibodies (mAbs) is performed using Protein A chromatography. We investigated the efficiency and robustness of cation exchange chromatography (CEX) in an effort to evaluate alternative capture methodologies. Up to five commercially available CEX resins were systematically evaluated using an experimentally optimized buffer platform and a design-of-experiment (DoE) approach for their ability to (a) capture a model mAb with a neutral isoelectric point, (b) clear three model viruses (porcine parvovirus, CHO type-C particles, and a bacteriophage). This approach identified a narrow operating space where yield, purity, and viral clearance were optimal under a CEX capture platform, and revealed trends between viral clearance of PPV and product purity (but not yield). Our results suggest that after unit operation optimization, CEX can serve as a suitable capture step. 相似文献
23.
Bailey MR Chen D Emery WR Lambooy PK Nolting J Quertinmont MT Shamlou PA 《Biotechnology and bioengineering》2008,99(6):1384-1391
Mammalian cell lines constitute an important part in the manufacture of therapeutic proteins. However, their susceptibility to virus contamination is a potential risk to patient safety and productivity, and has led to the development of a repertoire of virus inactivation techniques. From a process development viewpoint, the challenge is to demonstrate the required log reduction in virus content without a significant loss in product titer or quality. The balance between the two is dictated by the kinetics of virus inactivation and protein degradation, both of which are critically affected by process parameters. In this study we describe a commercially available microchannel reactor (MCR) and demonstrate how it can be used to evaluate the impact of temperature on the kinetics of virus inactivation and protein product degradation. Virus spiking experiments are reported using Xenotropic Murine Leukemia Virus and REOvirus, into buffers in the absence and presence of a therapeutic protein currently under development at Lilly. The results demonstrate that the MCR is an ideal platform for evaluation of fast reactive systems and reactions that are particularly sensitive to small changes to process conditions. These conditions include heat inactivation of a virus in a mammalian cell culture process stream used in the manufacture of therapeutic proteins and antibodies. 相似文献
24.
An automated microplate-based method for monitoring DNA strand breaks in plasmids and bacterial artificial chromosomes 下载免费PDF全文
A method is described for high-throughput monitoring of DNA backbone integrity in plasmids and artificial chromosomes in solution. The method is based on the denaturation properties of double-stranded DNA in alkaline conditions and uses PicoGreen fluorochrome to monitor denaturation. In the present method, fluorescence enhancement of PicoGreen at pH 12.4 is normalised by its value at pH 8 to give a ratio that is proportional to the average backbone integrity of the DNA molecules in the sample. A good regression fit (r2 > 0.98) was obtained when results derived from the present method and those derived from agarose gel electrophoresis were compared. Spiking experiments indicated that the method is sensitive enough to detect a proportion of 6% (v/v) molecules with an average of less than two breaks per molecule. Under manual operation, validation parameters such as inter-assay and intra-assay variation gave values of <5% coefficient of variation. Automation of the method showed equivalence to the manual procedure with high reproducibility and low variability within wells. The method described requires as little as 0.5 ng of DNA per well and a 96-well microplate can be analysed in 12 min providing an attractive option for analysis of high molecular weight vectors. A preparation of a 116 kb bacterial artificial chromosome was subjected to chemical and shear degradation and DNA integrity was tested using the method. Good correlation was obtained between time of chemical degradation and shear rate with fluorescence response. Results obtained from pulsed- field electrophoresis of sheared samples were in agreement with those obtained using the microplate-based method. 相似文献
25.
Mass balances were performed on an alkaline lysis operation for the primary recovery of supercoiled plasmid DNA as part of a process for plasmid gene preparation. Escherichia coli DH5alpha/pSVbeta was cultured in defined medium by fed-batch fermentation and harvested at the end of the exponential phase. Alkaline lysis of the recombinant cells was performed at fixed shear rates ranging between 46 and 461 s(-1), with neutralization 100 and 300 s after the initiation of the lysis. Mass balance calculations were used to optimize the operating conditions for carrying out the alkaline lysis operation. The results indicated that a plasmid yield of 75% and purity with respect to total DNA of 60% were achievable during the primary recovery operation. The influences of key contaminants, including the soluble proteins and the suspended solids, as they bear on the subsequent purification operations, were evaluated and discussed. 相似文献
26.
J. T. Tsai E. Keshavarz-Moore J. M. Ward M. Hoare P. Ayazi Shamlou P. Dunnill 《Bioprocess and biosystems engineering》1999,21(3):279-286
Plasmid DNA complexes have been used by several research groups for gene therapy applications and have given promising results during preliminary clinical trials. However, their eventual adoption for the treatment of a wide range of genetic diseases requires considerable progress with regards to carrier formulation and processing. Amongst the key parameters that are known to be important and need further elucidation are the size and surface charge of the conjugates and the binding efficiency of the carrier to the DNA. The present study has focused on two synthetic carriers with different formulation and charge characteristics, both of which have been recommended in the literature as having the potential to deliver genes to target cells. These were a cationic lipid (DC-chol/DOPE) and a polylysine-condensed anionic liposome (OA/DOPE/Chol). Conjugate size, zeta potential, stability and binding efficiency were measured for conjugates of DNA plasmids of molecular weights between 6.9 kb and 29 kb. Plasmids were condensed by polylysine of two different molecular weights with or without cationic lipids. 相似文献
27.
Engineering and cell attachment properties of human fibronectin–fibrinogen scaffolds for use in tissue engineered blood vessels 总被引:3,自引:0,他引:3
Harding SI Afoke A Brown RA MacLeod A Shamlou PA Dunnill P 《Bioprocess and biosystems engineering》2002,25(1):53-59
Tissue engineered constructs reported to date have been prepared primarily from poly(glycolic) acid or collagen scaffolds onto which cells are grown and matured. In this paper we report experimental data to demonstrate the use of a natural, human protein, as a tubular scaffold for vascular grafting. Using a manual and a scalable dip-coating technique we prepared fibronectin-based tubes up to 12 cm in length and up to 3 mm in diameter. The tubes were flexible and their mechanical properties, measured in terms of tensile strength and burst pressure as a function of humidity, demonstrated their suitability as scaffolds for use in vascular grafting, e.g. coronary artery by pass grafting. In vitro tests involved the attachment of endothelial cells pumped under laminar flow conditions through the tube lumen and the adherence of smooth muscle cells on the outer surface of the tubes. These tests, carried out in multiwells, showed that the scaffolds had excellent cell attachment and guidance characteristics. 相似文献
28.
Rheological properties of chromosomal and plasmid DNA during alkaline lysis reaction 总被引:1,自引:0,他引:1
L. A. S. Ciccolini P. Ayazi Shamlou N.J. Titchener-Hooker J. M. Ward P. Dunnill 《Bioprocess and biosystems engineering》1999,21(3):231-237
The experimental apparatus for the simultaneous L-lactic acid fermentation by Rhizopus oryzae immobilized in calcium alginate beads and product separation process was set up in which a three-phase fluidized-bed bioreactor was used as a fermentor and an external electrodialyzer as a separator, and a pump was applied to recycle the fermentation broth between the bioreactor and the separator. The L-lactic acid produced in the fermentor was separated in the separator, product inhibition was alleviated without any addition of alkali or alkali salts and the product purification process could be simplified. The specific productivity and the yield in electrodialysis fermentation (ED-F) process operated in continuous feeding mode were almost the same as that in CaCO3-buffered fermentation process. A mathematical model of L-lactic acid production in ED-F process was also suggested, in which the model equations for the bioreactor and the electrodialyzer were combined to describe the simultaneous fermentation and product separation. The model predictions were in good agreement with the experimental data. 相似文献
29.
Kirilynn Svay Christine Urrea Parviz Ayazi Shamlou Hu Zhang 《Biotechnology progress》2020,36(6):e3049
Single use bioreactors provide an attractive alternative to traditional deep-tank stainless steel bioreactors in process development and more recently manufacturing process. Wave bag bioreactors, in particular, have shown potential applications for cultivation of shear sensitive human and animal cells. However, the lack of knowledge about the complex fluid flow environment prevailing in wave bag bioreactors has so far hampered the development of a scientific rationale for their scale up. In this study, we use computational fluid dynamics (CFD) to investigate the details of the flow field in a 20-L wave bag bioreactor as a function of rocking angle and rocking speed. The results are presented in terms of local and mean velocities, mixing, and energy dissipation rates, which are used to create a process engineering framework for the scale-up of wave bag bioreactors. Proof-of-concept analysis of mixing and fluid flow in the 20-L wave bag bioreactor demonstrates the applicability of the CFD methodology and the temporal and spatial energy dissipation rates integrated and averaged over the liquid volume in the bag provide the means to correlate experimental volumetric oxygen transfer rates (kLa) data with power per unit volume. This correlation could be used as a rule of thumb for scaling up and down the wave bag bioreactors. 相似文献
30.
G. Aguilera Soriano N. J. Titchener-Hooker P. Ayazi Shamlou 《Bioprocess and biosystems engineering》1997,17(2):115-119
Experimental data are given for the solid pressure distributions in liquid chromatographic columns for two commercially available agarose-based compressible gel supports. The data show that for a given liquid velocity the pressure drop increases exponentially with height from the top surface. However, beyond a threshold liquid velocity, flow instability develops rapidly and the pressure drop rises sharply as the gel matrix compresses under the applied vertical pressures. The threshold liquid velocity at the point of criticality is examined in the light of the experimental data and the numerical simulations based on a one dimensional elemental slice force balance of the gel mass. The result of this analysis shows that matrix rigidity, column diameter and bed height significantly affect the point of criticality. 相似文献