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Chromatography Column (chromatography + column)
Selected AbstractsCharacterization of Herpes Simplex Virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activityPROTEIN SCIENCE, Issue 9 2002Mark S. Kokoris Abstract Herpes Simplex Virus type 1 (HSV-1) thymidine kinase (TK) is currently the most widely used suicide agent for gene therapy of cancer. Tumor cells that express HSV-1 thymidine kinase are rendered sensitive to prodrugs due to preferential phosphorylation by this enzyme. Although ganciclovir (GCV) is the prodrug of choice for use with TK, this approach is limited in part by the toxicity of this prodrug. From a random mutagenesis library, seven thymidine kinase variants containing multiple amino acid substitutions were identified on the basis of activity towards ganciclovir and acyclovir based on negative selection in Escherichia coli. Using a novel affinity chromatography column, three mutant enzymes and the wild-type TK were purified to homogeneity and their kinetic parameters for thymidine, ganciclovir, and acyclovir determined. With ganciclovir as the substrate, one mutant (mutant SR39) demonstrated a 14-fold decrease in Km compared to the wild-type enzyme. The most dramatic change is displayed by mutant SR26, with a 124-fold decrease in Km with acyclovir as the substrate. Such new "prodrug kinases" could provide benefit to ablative gene therapy by now making it feasible to use the relatively nontoxic acyclovir at nanomolar concentrations or ganciclovir at lower, less immunosuppressive doses. [source] High-throughput quantification of selenium in individual serum proteins from a healthy human population using HPLC on-line with isotope dilution inductively coupled plasma-MSPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 19 2010Sophia Letsiou Abstract In this study, a method, based on dual column affinity chromatography hyphenated to isotope dilution inductively coupled plasma,quadrupole MS, was developed for selenium determination in selenoprotein P, glutathione peroxidase, and selenoalbumin in human serum samples from a group of healthy volunteers (n=399). Method improvement was achieved using methanol-enhanced isotope dilution which resulted in improved sensitivity and removal of isobaric interferences. Although no human serum reference materials are currently certified for their selenium species levels, method development was conducted using human serum reference material BCR 637 and 639 as their Se species content has been reported in the previous studies, and thus comparisons were possible. The mean selenium concentrations determined for the 399 healthy volunteer serum samples were 23±10,ng Se mL,1 for glutathione peroxidase, 49±15,ng Se mL,1 for selenoprotein P and 11±4,ng Se mL,1 for selenoalbumin. These values are found to be in close agreement with published values for a limited number of healthy volunteer samples, and to establish baseline Se levels in serum proteins for an apparently healthy group of individuals, thus allowing for subsequent comparisons with respective values determined for groups of individuals with selenium related health issues, as well as assist in the discovery of potential selenium biomarkers. Also, the relationship between Se serum protein levels and some anthropometric characteristics of the volunteer population were investigated. Additionally, further development of the analytical method used in this study was achieved by adding a size exclusion chromatography column after the two affinity columns via a switching valve. This allowed for the separation of small selenium-containing molecules from glutathione peroxidase and thus enhanced the overall confidence in its identification. [source] Method optimisation for peptide profiling of microdissected breast carcinoma tissue by matrix-assisted laser desorption/ionisation-time of flight and matrix-assisted laser desorption/ionisation-time of flight/time of flight-mass spectrometryPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 10 2005Arzu Umar Abstract Appropriate methods for the analysis of microdissected solid tumour tissues by matrix-assisted laser desorption/ionisation-time of flight-mass spectrometry (MALDI-TOF MS) are not yet well established. Optimisation of sample preparation was performed first on undissected tissue slices, representing ,200,000 cells, which were solubilised either in urea containing buffer, trifluoroethanol/NH4HCO3, 0.1% sodium dodecyl sulphate (SDS) or in 0.1% RapiGest solution, then trypsin digested and analysed by MALDI-TOF MS. Solubilisation in 0.1% SDS resulted in detection of the highest number of sample specific peak signals. Interestingly, there was little overlap in detectable peaks using the different buffers, implying that they can be used complementarily to each other. Additionally, we fractionated tryptic digests on a monolithic high-performance liquid chromatography column. Fractionation of tryptic digest from whole tissue sections resulted in a four-fold increase in the total number of peaks detected. To prove this principle, we used 0.1% SDS to generate peptide patterns from 2000 microdissected tumour and stromal cells from five different breast carcinoma tumours. The tumour and stroma specific peaks could be detected upon comparison of the peptide profiles. Identification of differentially expressed peaks by MALDI-TOF/TOF MS was performed on fractionated tryptic digests derived from a whole tissue slice. In conclusion, we describe a method that is suitable for direct peptide profiling on small amounts of microdissected cells obtained from breast cancer tissues. [source] Gold-coated fused-silica sheathless electrospray emitters based on vapor-deposited titanium adhesion layersRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 14 2003Stefan Nilsson Gold-coated fused-silica electrospray (ES) emitters based on vapor-deposited adhesion layers of titanium have been manufactured to investigate the possibilities of producing durable ES emitters applicable in chip-based analytical devices. The stabilities of the emitters were studied by both electrospray and electrochemical experiments and a marked increase in the emitter lifetime, compared to that for Cr/Au coated emitters, was found for the Ti/Au emitters in the ES durability tests. This indicates that Ti (rather than Cr) adhesion layers should be used in association with large-scale fabrication of ES emitters by vapor-deposition techniques. The lifetime of about 500,700 hours also allowed the Ti/Au-coated emitter to be used as an integrated part of a capillary liquid chromatography column coupled to a mass spectrometer in a series of LC/MS experiments. The Ti/Au coating was further studied by electrochemical techniques and scanning electron microscopy in conjunction with X-ray spectroscopy. It is shown that the eventual failure of the Ti/Au emitters in ES experiments was due to an almost complete detachment of the gold layer. Experimental evidence suggests that the detachment of the gold coating was due to a reduced adhesion to the titanium layer during oxidation in positive electrospray. Most likely, this was caused by the formation of an oxide layer on the titanium film. It is thus shown that unlimited emitter stabilities are not automatically obtained even if the metallic adhesion layer is stabilized by an oxide formation under positive electrospray conditions. Copyright © 2003 John Wiley & Sons, Ltd. [source] Preparation of a super-long two column chromatography system and its application in separating glycosylated puerarinBIOMEDICAL CHROMATOGRAPHY, Issue 12 2009Shouchuang Zhu Abstract Separation of Puerarin-7- O -glucoside from its precursor, puerarin, using a common chromatography column packed with AB-8 macroporous resin was unsuccessful. Therefore, in this study a 8,m super-long flexible reinforced PVC column was externally added to the common column in order to improve the chromatography efficiency by increasing the number of theoretical plates. Both the PVC and common columns were separately packed with AB-8 macroporous resin slurry. The packed PVC column was coiled after washing and stored until use. The microbial transformation mixture with puerarin-7- O -glucoside and puerarin (250,mL) was loaded onto the common column, followed by washing with 2000,mL H2O. After attaching the coiled external PVC column to the common column, a linear gradient of 10,30% ethanol was applied to elute the target compound. Two peaks appeared: peak I contained puerarin-7- O -glucoside at 97.9% purity and 88.1% recovery rate, and peak II was puerarin at 98.7% purity and 87.0% recovery rate. The use of the coiled external flexible reinforced PVC column avoided spatial restriction for long columns, which made it much more convenient for column packing and chromatography operations. Furthermore, this method eliminated the resin blockage problem caused by stationary water pressure in a rigid vertical long column. Using an external super-long column, the PVC tube was connected with the common column only during elution, which avoided delay in time period during sample loading and column washes associated with the use of long external columns. Copyright © 2009 John Wiley & Sons, Ltd. [source] Dynamics of protein uptake within the adsorbent particle during packed bed chromatographyBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2002Jürgen Hubbuch Abstract A new experimental set-up for on-line visualization of the intra-particle uptake kinetics during packed bed chromatography has been designed and tested. Confocal laser scanning microscopy was used to analyze the dynamics of protein adsorption to porous stationary phases. In combination with this, a flow cell was developed that could be packed with chromatography media and operated as a fully functional mini-scale chromatography column. Adsorption profiles of single- and two-component mixtures containing BSA and IgG 2a during packed bed cation-exchange chromatography were investigated. The two proteins appear to exhibit different transport characteristics. For BSA a classical "shrinking core" behavior could be detected. The profiles obtained during IgG 2a adsorption point toward a different transport mode, which deviates from the classical pore-diffusion picture. For the two-component system, a superposition of the single-component profiles combined with a classical displacement of the weaker bound protein species was found. The results indicate that depending on the adsorbed protein the uptake can vary tremendously, even for adsorption to the same chromatographic support. It is clearly shown that the new microcolumn allows in situ quantitative investigations of protein adsorption dynamics within a single particle, which adds a new tool to the available methods for characterizing and optimizing protein adsorption chromatography. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 359,368, 2002. [source] Large scale demonstration of a process analytical technology application in bioprocessing: Use of on-line high performance liquid chromatography for making real time pooling decisions for process chromatographyBIOTECHNOLOGY PROGRESS, Issue 2 2010Anurag S. Rathore Abstract Process Analytical Technology (PAT) has been gaining a lot of momentum in the biopharmaceutical community because of the potential for continuous real time quality assurance resulting in improved operational control and compliance. In previous publications, we have demonstrated feasibility of applications involving use of high performance liquid chromatography (HPLC) and ultra performance liquid chromatography (UPLC) for real-time pooling of process chromatography column. In this article we follow a similar approach to perform lab studies and create a model for a chromatography step of a different modality (hydrophobic interaction chromatography). It is seen that the predictions of the model compare well to actual experimental data, demonstrating the usefulness of the approach across the different modes of chromatography. Also, use of online HPLC when the step is scaled up to pilot scale (a 2294 fold scale-up from a 3.4 mL column in the lab to a 7.8 L column in the pilot plant) and eventually to manufacturing scale (a 45930 fold scale-up from a 3.4 mL column in the lab to a 158 L column in the manufacturing plant) is examined. Overall, the results confirm that for the application under consideration, online-HPLC offers a feasible approach for analysis that can facilitate real-time decisions for column pooling based on product quality attributes. The observations demonstrate that the proposed analytical scheme allows us to meet two of the key goals that have been outlined for PAT, i.e., "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions". The application presented here can be extended to other modes of process chromatography and/or HPLC analysis. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Preparing titania aerogel monolithic chromatography columns using supercritical carbon dioxideJOURNAL OF SEPARATION SCIENCE, JSS, Issue 11 2010Ruohong Sui Abstract The search for a method to fabricate monolithic inorganic columns has attracted significant recent attention due to their unique ability in separation applications of various biomolecules. Silica and polymer based monolithic columns have been prepared, but titania and other metal oxide monoliths have been elusive, primarily due to their fragility. This article describes a new approach for preparing nanostructured titania based columns, which offer better performance over conventional particle packed columns for separating a wide variety of biomolecules including phosphopeptides. TiO2 monolithic aerogels were synthesized in separation columns using in situ sol-gel reactions in supercritical carbon dioxide (scCO2) followed by calcination, and compared to those prepared in heptanes. The characterization results show that scCO2 is a better solvent for the sol-gel reactions, providing lower shrinkage with the anatase TiO2 monolith composed of nanofibers with very high surface areas. The monolithic columns show the ability to isolate phosphopeptides with little flow resistance compared to conventional titania particle based microcolumns. [source] An evaluation of the robustness of the Tanaka characterization protocol for reversed-phase liquid chromatography columnsJOURNAL OF SEPARATION SCIENCE, JSS, Issue 16 2005Patrik Petersson Abstract The robustness of the popular ,Tanaka' LC column characterization protocol has been evaluated by the statistical tools of reduced factorial design, multiple linear regression and principal component analysis. These have shown that in order to obtain reliable and reproducible results, it is especially important to control the methanol content, the temperature and, in the case of the total ion-exchange capacity test (,B/P pH 7.6), the pH of the mobile phase. In particular, the hydrophobicity tests (kPB and ,) are sensitive to small changes in methanol content. Provided that the operating parameters for the Tanaka column characterization protocol are controlled within the following experimental limits, i. e. methanol content ±0.5% v/v, temperature ±3°C, pH ±0.10 and buffer concentration ±2.0 mM, it is feasible to distinguish between RP materials that possess selectivity differences larger than their batch-to-batch reproducibility. These experimental requirements can be easily met by current LC instrumentation. Hence, the validity of the Tanaka testing protocol for characterizing columns has been verified. [source] Glutathione S-transferases in the adaptation to plant secondary metabolites in the Myzus persicae aphidARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2005Frédéric Francis Abstract Glutathione S-transferases (GST) in insects play an important role in the detoxification of many substances including allelochemicals from plants. Induction of GST activity in Myzus persicae in response to secondary metabolites from Brassica plants was determined using different host plant species and confirmed using artificial diet with pure allelochemicals added. The 2,4-dinitro-1-iodobenzene (DNIB) was found to be a useful substrate for identifying particular GSTs in insects. GSTs from M. persicae were purified using different affinity chromatography columns and related kinetic parameters were calculated. GST isoenzymes were characterised using electrophoretic methods. Although SDS-PAGE results indicated similarity among the purified enzymes from each affinity column, biochemical studies indicated significant differences in kinetic parameters. Finally, the GST pattern of M. persicae was discussed in terms of insect adaptation to the presence of plant secondary substances such as the glucosinolates and the isothiocyanates, from Brassicaceae host plants. Arch. Insect Biochem. Physiol. 58:166,174, 2005. © 2005 Wiley-Liss, Inc. [source] Case study and application of process analytical technology (PAT) towards bioprocessing: Use of tryptophan fluorescence as at-line tool for making pooling decisions for process chromatographyBIOTECHNOLOGY PROGRESS, Issue 5 2009Anurag S. Rathore Abstract Process analytical technology (PAT) has been gaining momentum in the biopharmaceutical community due to the potential for continuous real time quality assurance resulting in improved operational control and compliance. Two imperatives for implementing any PAT tool are that "variability is managed by the process" and "product quality attributes can be accurately and reliably predicted over the design space established for materials used, process parameters, manufacturing, environmental, and other conditions." Recently, we have been examining the feasibility of applying different analytical tools to bioprocessing unit operations. We have previously demonstarted that commercially available online-high performance liquid chromatography and ultra performance liquid chromatography systems can be used for analysis that can facilitate real-time decisions for column pooling based on product quality attributes (Rathore et al., 2008a,b). In this article, we review an at-line tool that can be used for pooling of process chromatography columns. We have demonstrated that our tryptophan fluorescence method offers a feasible approach and meets the requirements of a PAT application. It is significantly faster than the alternative of fractionation, offline analysis followed by pooling. Although the method as presented here is not an online method, this technique may offer better resolution for certain applications and may be a more optimal approach as it is very conducive to implementation in a manufacturing environment. This technique is also amenable to be used as an online tool via front face fluorescence measurements done concurrently with product concentration determination by UV. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Ultra scale-down approach to correct dispersive and retentive effects in small-scale columns when predicting larger scale elution profilesBIOTECHNOLOGY PROGRESS, Issue 4 2009N. Hutchinson Abstract Ultra scale-down approaches represent valuable methods for chromatography development work in the biopharmaceutical sector, but for them to be of value, scale-down mimics must predict large-scale process performance accurately. For example, one application of a scale-down model involves using it to predict large-scale elution profiles correctly with respect to the size of a product peak and its position in a chromatogram relative to contaminants. Predicting large-scale profiles from data generated by small laboratory columns is complicated, however, by differences in dispersion and retention volumes between the two scales of operation. Correcting for these effects would improve the accuracy of the scale-down models when predicting outputs such as eluate volumes at larger scale and thus enable the efficient design and operation of subsequent steps. This paper describes a novel ultra scale-down approach which uses empirical correlations derived from conductivity changes during operation of laboratory and pilot columns to correct chromatographic profiles for the differences in dispersion and retention. The methodology was tested by using 1 mL column data to predict elution profiles of a chimeric monoclonal antibody obtained from Protein A chromatography columns at 3 mL laboratory- and 18.3 L pilot-scale. The predictions were then verified experimentally. Results showed that the empirical corrections enabled accurate estimations of the characteristics of larger-scale elution profiles. These data then provide the justification to adjust small-scale conditions to achieve an eluate volume and product concentration which is consistent with that obtained at large-scale and which can then be used for subsequent ultra scale-down operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Mechanical Deformation of Compressible Chromatographic ColumnsBIOTECHNOLOGY PROGRESS, Issue 3 2002R. N. Keener A one-dimensional model of mechanical deformation of compressible chromatography columns is presented. The model is based on linear elasticity and continuum mechanics and is compared to a more complete two-dimensional model and one-dimensional porosity profiles measured by NMR imaging methods. The model provides a quantitative description of compression and the effects of wall support during scale-up. A simple criterion for the significance of wall support as a function of both diameter and length is also developed. Although the model accounts only for mechanical deformation, flow compression can be included, and validation presented here suggests that a more complete model may be valuable for anticipating the effects of scale and aspect ratio on pressure-flow behavior of compressible columns. [source] | |||||||||||||