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Gradient Chromatography (gradient + chromatography)
Selected AbstractsSurface-activated chemical ionization and high-flow gradient chromatography to reduce matrix effectRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 16 2006Simone Cristoni The new atmospheric pressure chemical ionization source, named surface-activated chemical ionization (SACI), has been used in conjunction with high-flow gradient chromatography to reduce the matrix effect. This high-flow gradient chromatography approach avoids the co-elution of analyte and biological matrix compounds that leads to a reduction in quantitation errors due to matrix effect. However, this approach cannot be employed with the classical electrospray ionization (ESI) source that usually works at low eluent flow (<,300,µL/min). SACI can work at high eluent flow (100,2000,µL/min) and can be employed in conjunction with high-flow gradient chromatography. The reduction in matrix effect in tacrolimus analysis in protein-precipitated blood samples, an important immunosuppressive agent for renal transplantation, is presented and discussed. Copyright © 2006 John Wiley & Sons, Ltd. [source] A rapid and simple HPLC-UV method for the determination of inhibition characteristics of farnesyl transferase inhibitorsBIOMEDICAL CHROMATOGRAPHY, Issue 2 2006Natalie M. G. M. Appels Abstract Ras proteins play an important role in the development of cancer. Farnesyl transferase inhibitors (FTIs) block the first obligatory post-translational step for activation, prenylation, of Ras proteins. To find new potent FTIs, rapid enzyme activity assays are required to reduce FTI development time. Most assays to date are based on radioactive labelled substrates. We developed a new, in vitro, farnesyl transferase assay based on gradient chromatography coupled to UV detection. Unfarnesylated and farnesylated H-Ras proteins were resolved on a C18 wide-pore HPLC column and their concentrations were determined with use of a calibration curve of unfarnesylated H-Ras. The assay was used to investigate inhibition characteristics of FTIs. The IC50 values of the FTIs L778,123 and SCH66336 were 4.2 nm and 78 µm, respectively. This assay could support the screening and development of FTIs to obtain rapid insights into their inhibitory properties. Copyright © 2005 John Wiley & Sons, Ltd. [source] Increasing the activity of monoclonal antibody therapeutics by continuous chromatography (MCSGP)BIOTECHNOLOGY & BIOENGINEERING, Issue 4 2010T. Müller-Späth Abstract The charged monoclonal antibody (mAb) variants of the commercially available therapeutics Avastin®, Herceptin® and Erbitux® were separated by ion-exchange gradient chromatography in batch and continuous countercurrent mode (MCSGP process). Different stationary phases, buffer conditions and two MCSGP configurations were used in order to demonstrate the broad applicability of MCSGP in the field of charged protein variant separation. Batch chromatography and MCSGP were compared with respect to yield, purity, and productivity. In the case of Herceptin®, also the biological activity of the product stream was taken into account as performance indicator. The robustness of the MCSGP process against feed composition variations was confirmed experimentally and by model simulations. Biotechnol. Bioeng. 2010;107:652,662. © 2010 Wiley Periodicals, Inc. [source] Investigation of protein binding affinity and preferred orientations in ion exchange systems using a homologous protein libraryBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2009Wai Keen Chung Abstract A library of cold shock protein B (CspB) mutant variants was employed to study protein binding affinity and preferred orientations in cation exchange chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography. While the expected trends were observed with increasing or decreasing positive charge on the protein surface, the degree of change was a strong function of the location and microenvironment of the mutated amino acid. Quantitative structure,property relationship (QSPR) models were generated using a support vector regression technique that was able to give good predictions of the retention times of the various mutants. Molecular descriptors selected during model generation were used to elucidate the factors affecting protein retention. Electrostatic potential maps were also employed to provide insight into the effects of protein surface topography, charge density and charge distribution on protein binding affinity and possible preferred binding orientations. The use of this protein mutant library in concert with the qualitative and quantitative analyses presented in the article provides an improved understanding of protein behavior in ion exchange systems. Biotechnol. Bioeng. 2009; 102: 869,881. © 2008 Wiley Periodicals, Inc. [source] | ||||||||||