The Experts below are selected from a list of 183 Experts worldwide ranked by ideXlab platform
Douglas D Frey - One of the best experts on this subject based on the ideXlab platform.
-
evaluation of Chromatofocusing as a capture method for monoclonal antibody products
Journal of Chromatography A, 2018Co-Authors: Yang Liu, Sevda Deldari, Hui Guo, Chittoor R Narahari, Ronald Bates, Ryan K Swanson, Sanchayita Ghose, Douglas D FreyAbstract:Chromatofocusing is investigated as an alternative to protein A chromatography for the initial capture step in a purification process for several monoclonal antibodies and antibody fusion products. For comparison, this work also investigates the use of ion-exchange chromatography with either pH or salt gradient elution as additional alternatives to protein A chromatography. The specific conditions employed for the capture step for the case of Chromatofocusing were selected on a rational basis using a computer-aided design method implemented in the form of a Microsoft Excel spreadsheet. Alternative operating conditions were compared experimentally with regard to the product yield achieved as well as the removal of total host cell proteins (HCPs) and of a specific HCP major component. Results from this study indicate that both Chromatofocusing and ion-exchange chromatography are useful alternatives to a protein A chromatography capture step in many practical cases. This is especially true for the case of Chromatofocusing when it is possible to exploit the ability of the method to create complex gradient shapes that are self-forming inside the column and to simultaneous focus and separate proteins inside the column.
-
development of Chromatofocusing techniques employing mixed mode column packings for protein separations
Journal of Chromatography A, 2014Co-Authors: Hui Guo, Douglas D FreyAbstract:Recent studies reported in the literature using mixed-mode chromatography (MMC) column packings have shown that multiple modes of interactions between the column packing and proteins can be usefully exploited to yield excellent resolution as well as salt-tolerant adsorption of the target protein. In this study, a mixed-mode separation method using commercially available column packings was explored which combines the techniques of hydrophobic-interaction chromatography and Chromatofocusing. Two different column packings, one based on mercapto-ethyl-pyridine (MEP) and the other based on hexylamine (HEA) were investigated with regard to their ability to separate proteins when using internally generated, retained pH gradients. The effects of added salt and urea on the behavior of the retained pH gradient and the protein separation achieved when using MMC column packings for Chromatofocusing were also investigated. Numerical simulations using methods developed in previous work were shown to agree with experimental results when using reasonable physical parameters. These numerical simulations were also shown to be a useful qualitative method to select the compositions of the starting and elution buffers in order to achieve desired shapes for the pH and ionic strength gradients. The use of the method to fractionate blood serum was explored as a prototype example application.
-
characterization and purification of bacteriophages using Chromatofocusing
Journal of Chromatography A, 2008Co-Authors: Kurt Brorson, Hong Shen, Scott Lute, Jessica Soto Perez, Douglas D FreyAbstract:The technique of Chromatofocusing was applied to the characterization and purification of three bacteriophages that are routinely used for testing virus filters: φX174, PR772, and PP7. Chemically well-defined eluent buffers were used, instead of the more commonly used Chromatofocusing polyampholyte buffers. Chromatographic column packings were selected to minimize band broadening by confining bacteriophage adsorption solely to the exterior particle surface. Under the conditions used it was determined that bacteriophages could be made to focus into narrow bands in a retained pH gradient with recoveries of live phage that ranged from 15 to nearly 100% as determined by a plaque-forming assay. Retention times and apparent isoelectric point data were obtained for samples consisting either of purified bacteriophage, or samples consisting of crude preparations of bacteriophages containing host cell impurities. Isoelectric point estimates were obtained using modified, previously described models. The results obtained suggest that Chromatofocusing is a simple and rapid method for obtaining approximate isoelectric points for bacteriophages and probably other types of viruses. It is also likely a useful method for purifying these materials.
-
reducing sample complexity in proteomicsby Chromatofocusing with simple buffer mixtures
Methods of Molecular Biology, 2008Co-Authors: Hong Shen, Charles J Bieberich, Douglas D FreyAbstract:Chromatofocusing has many potential applications in the field of proteomics, such as for the isolation and removal of major sample components to facilitate the analysis of low-abundance components, and for sample prefractionation prior to a subsequent separation using SDS-PAGE, narrow-pI-range 2D-PAGE, or additional chromatography steps. However, the Chromatofocusing techniques that are most commonly used employ propriety polyampholyte elution buffers and highly specialized column packings, both of which limit the use of Chromatofocusing in practice. To expand the range of application for this technique, this chapter considers Chromatofocusing methods which employ common ion-exchange column packings and elution buffers which are simple mixtures of readily available buffering species. Of particular interest is the use of Chromatofocusing with a multistep pH gradient for the fractionation of protein mixtures into narrow-pI-range fractions. The cross-contamination characteristics of these fractions using SDS-PAGE are also assessed.
-
Chromatofocusing of peptides and proteins using linear ph gradients formed on strong ion exchange adsorbents
Biotechnology and Bioengineering, 2004Co-Authors: Xuezhen Kang, Douglas D FreyAbstract:Although it is commonly believed that a column packing used for Chromatofocusing must have an "even" buffering capacity in order to produce a linear pH gradient, it is demonstrated here that linear pH gradients suitable for Chromatofocusing can be produced on a column packing having a minimal buffering capacity. In particular, if either a strong-acid cation-exchange column packing or a strong-base anion-exchange column packing is presaturated with either a weak acid titrated with a strong base, or a weak base titrated with a strong acid, respectively, to the initial pH, then a linear or nearly linear pH gradient can be formed using a polyampholyte elution buffer by taking advantage of the presence of small quantities of weak-acid or weak-base functional groups that generally exist on these types of column packings. Experimental and theoretical studies are used to demonstrate that such systems have potential advantages over traditional Chromatofocusing methods in terms of the speed of the separation, the resolution achieved, and the range of applications possible. Among other techniques described, a method for separating tryptic peptides using Chromatofocusing and a strong-acid cation-exchange column packing is demonstrated to be a useful alternative to capillary isoelectric focusing and ion-exchange chromatography using a salt gradient for this purpose.
H D Bartunik - One of the best experts on this subject based on the ideXlab platform.
-
purification and crystallization of yeast hexokinase isoenzymes characterization of different forms by Chromatofocusing
Journal of Chromatography A, 1991Co-Authors: L R Jacob, V Beecken, L J Bartunik, M Rose, H D BartunikAbstract:The yeast hexokinase isoenzymes PI and PII have been purified in large amounts (20 mg) from overproducing yeast strains. The purification procedures of hexokinase PI and PII include anion-exchange chromatography on DEAE-Sephacel and Chromatofocusing on PBE 94, hydrophobic interaction chromatography on phenyl-Sepharose (necessary for the isolation of the isoenzyme PI); in the final step either a Mono Q HR 5/5 or a Fractogel EMD TMAE 650(S) column was used. Hexokinase preparations were characterized before crystallization by Chromatofocusing on a Mono P HR 5/20 FPLC column, where different forms of hexokinase can be rapidly distinguished by their elution behaviour. From both purified hexokinase PI and PII, large crystals were grown that diffract X-rays to high resolution.
David J Anderson - One of the best experts on this subject based on the ideXlab platform.
-
Gradient Chromatofocusing-mass spectrometry: A new technique in protein analysis
Journal of the American Society for Mass Spectrometry, 2008Co-Authors: Lian Shan, James A. Hribar, Xiang Zhou, David J AndersonAbstract:A new analytical technique, gradient Chromatofocusing-mass spectrometry (gCF-MS), was developed employing ion-exchange high-performance liquid chromatography (HPLC) interfaced to an electrospray-quadrupole mass spectrometer in the determination of proteins. There have been few reports, if any, of a HPLC-MS technique for proteins in which the ion-exchange column is directly interfaced to the mass spectrometer. The employment of a linear pH gradient elution scheme directly interfaced to mass spectrometry is also unique in the present work. The technique was demonstrated by the separation of six proteins (carbonic anhydrase II, enolase, β-lactoglobulin A, lactoglobulin B, soybean trypsin inhibitor, and amyloglucosidase) employing a descending linear pH gradient from pH 9 to 2.6 on a 50 mm × 2.1 mm DEAE HPLC column using volatile buffer components. A signal enhancement solution consisting of 8% formic acid in acetonitrile was pumped post-column and was mixed 1:1 with column effluent and then directed on-line into the mass spectrometer. Molecular masses of the proteins were determined within ±0.010% to 0.033% (±100 to 330 ppm) with peak height total ion current detection limits of 4 to 78 pmol of injected amounts ( S/N =3). This technique is applicable to the analysis of proteins and other charged molecules.
-
gradient Chromatofocusing versatile ph gradient separation of proteins in ion exchange hplc characterization studies
Analytical Chemistry, 2002Co-Authors: Lian Shan, David J AndersonAbstract:A new Chromatofocusing technique called gradient Chromatofocusing is characterized. Gradient Chromatofocusing generates linear pH gradients on anion-exchange columns with inexpensive low molecular mass buffer components via HPLC gradient mixing. Gradient Chromatofocusing results are compared with that of conventional Chromatofocusing in the chromatography of several proteins on a Mono P column, including beta-lactoglobulin A and B, ovalbumin, BSA, and conalbumin. Gradient Chromatofocusing shows superior performance, with resolution increases greater than 3-fold being realized for the entire protein mixture and up to 25-fold for a particular protein pair. This performance superiority arises from inherent advantages in the gradient Chromatofocusing technique in optimizing conditions pertinent to separation, including buffer concentration and pH gradient slope. These resolution gains arise from both increases in separation factor and decreases in peak width achieved with the pH gradient Chromatofocusing technique through the manipulation of buffer concentration and the pH gradient profile. Gradient Chromatofocusing is also compared with conventional NaCl gradient ion-exchange chromatography using the same Mono P column, demonstrating 3-fold resolution gains, resulting from a 3-fold decrease in peak width. The present work demonstrates the significantly improved performance that gradient Chromatofocusing has in protein separations compared to other ion-exchange chromatographic techniques. Mechanisms for the various effects are discussed.
-
effect of buffer concentration on gradient Chromatofocusing performance separating proteins on a high performance deae column
Journal of Chromatography A, 2001Co-Authors: Lian Shan, David J AndersonAbstract:Gradient Chromatofocusing is a recently developed chromatographic technique that overcomes the limitations of conventional Chromatofocusing. This technique employs a HPLC gradient system and simple low-molecular-mass buffer components to generate linear or other function pH gradients on ion-exchange columns. Results of the present work show a superior separation of beta-lactoglobulin A and B in gradient Chromatofocusing compared to salt gradient chromatography using the same DEAE column, with an optimized resolution of 2.3 obtained with gradient Chromatofocusing compared to 1.1 obtained with NaCl gradients at constant pH. A significant advantage of the gradient Chromatofocusing technique over the conventional Chromatofocusing technique is its ability to employ a relatively wide range of buffer concentrations in the mobile phase, the effect of which is studied in the present work. Five proteins (conalbumin, ovalbumin, bovine serum albumin, beta-lactoglobulin A and B) are chromatographed on a DEAE-polymethacrylate HPLC anion-exchange column using the same approximately linear pH gradient profile but different mobile phase buffer concentrations. Results show a significant effect of buffer concentration on peak width, separation factor and resolution. For example, resolution increases from 1.5 to 2.3 in the separation of beta-lactoglobulin A and B when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 25.0 mM (with the same outlet pH gradient). This separation trend is also seen in the chromatography of ovalbumin from a commercial source, noting a progressive increase in resolution of two peaks in the sample (resolution increased from 0.7 to 2.4) when the concentration of each of the components in the 100% elution buffer is increased from 6.25 to 37.5 mM (same outlet pH gradient). The gains in the resolution are attributed to an increase in the separation factor, since the peak widths are generally noted to also increase with increased buffer concentration. These results point to a significant interplay between buffer concentration and pH, which is not effectively exploited in either conventional Chromatofocusing or in conventional ion-exchange chromatographic procedures employing salt gradient elution at constant pH. Gradient Chromatofocusing has the ability of optimizing both parameters, thus providing it with unique capabilities in protein separations.
-
gradient Chromatofocusing high performance liquid chromatography i practical aspects
Journal of Chromatography A, 1997Co-Authors: Yansheng Liu, David J AndersonAbstract:In this work, a versatile method for generating linear pH gradients using weak anion-exchange HPLC has been developed, which is termed gradient Chromatofocusing high-performance liquid chromatography. This method utilizes a linear external pH gradient generated in the mobile phase entering the column (inlet pH gradient), superimposed on an internally-generated pH gradient within the column (column pH gradient), which results from the buffering action of the ion exchanger on the mobile phase and vice versa. The method shows significant advantages over conventional Chromatofocusing, including: decreased expense due to the use of common buffer components, ease of adjusting the slope of the pH gradient produced at the outlet of the column (outlet pH gradient) through the manipulation of the inlet pH gradient and the ability of using high concentration buffers in the mobile phase. Chromatography of fibrinogen degradation products was done using gradient Chromatofocusing. Bandwidths comparable to conventional Chromatofocusing were obtained in the separation of fibrinogen degradation products.
L R Jacob - One of the best experts on this subject based on the ideXlab platform.
-
purification and crystallization of yeast hexokinase isoenzymes characterization of different forms by Chromatofocusing
Journal of Chromatography A, 1991Co-Authors: L R Jacob, V Beecken, L J Bartunik, M Rose, H D BartunikAbstract:The yeast hexokinase isoenzymes PI and PII have been purified in large amounts (20 mg) from overproducing yeast strains. The purification procedures of hexokinase PI and PII include anion-exchange chromatography on DEAE-Sephacel and Chromatofocusing on PBE 94, hydrophobic interaction chromatography on phenyl-Sepharose (necessary for the isolation of the isoenzyme PI); in the final step either a Mono Q HR 5/5 or a Fractogel EMD TMAE 650(S) column was used. Hexokinase preparations were characterized before crystallization by Chromatofocusing on a Mono P HR 5/20 FPLC column, where different forms of hexokinase can be rapidly distinguished by their elution behaviour. From both purified hexokinase PI and PII, large crystals were grown that diffract X-rays to high resolution.
Brian C S Liu - One of the best experts on this subject based on the ideXlab platform.
-
Chromatofocusing fractionation and two dimensional difference gel electrophoresis for low abundance serum proteins
Proteomics, 2005Co-Authors: Shuzhen Qin, Angeline Ferdinand, Jerome P Richie, Michael P Oleary, Samuel C Mok, Brian C S LiuAbstract:The technical challenge to analysis of the serum proteome is that the serum proteins are present at unequal concentrations. A few are so dominant, such as serum albumin and immunoglobulins, that they mask detection of other proteins. Because of these high abundance proteins, current technologies, while theoretically capable of analyzing protein amounts spanning four orders of magnitude, are only able to analyze proteins ranging over two orders of magnitude and cannot analyze the lower abundance proteins that may be the next biomarkers and drug targets. To facilitate the identification of low abundance proteins, we fractionated serum samples from patients with prostate cancer and patients with benign prostate hyperplasia using anion displacement liquid Chromatofocusing chromatography, which separates proteins by a pH gradient and a positively charged column. Differential expression of proteins from fractions was then determined and identified by IEF gels and 2-D DIGE. Results demonstrate improved resolution of proteins within the chosen pH gradient when compared to the unfractionated samples. Several proteins that were differentially expressed in serum from patients with prostate cancer were identified in the fractionated serum. Three of these proteins, squamous cell carcinoma antigen 1 (SCCA1), calgranulin B, and haptoglobin-related protein, are present in the serum at levels below the classical protein level of mg/mL. SCCA1 is normally expressed in serum at ng/mL levels, and calgranulin B is an intracellular protein. Our results demonstrate that the use of anion displacement liquid Chromatofocusing chromatography may reduce the complexity of the serum proteome by separating proteins into distinct pH ranges, and facilitate the identification of low abundance proteins.