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ABSTRACT: Proteins in bile may have important physiological functions and serve as disease biomarkers. Here, the protein composition of human gallbladder bile was analyzed using a recently described chromatography-like technology capable to enhance the signal of low-abundance species. First, proteins present in bile fluid were treated with immobilized peptide ligand libraries to concentrate dilute and very dilute species while concomitantly diluting the high-abundance proteins. The analysis of resulting protein mixture was then performed using LC-MS/MS after having classically separated proteins by a mini preparative gel electrophoresis. Overall 222 gene products were found; 143 of them were not reported before in proteomics studies. Ligand libraries by themselves contributed to find 81 new gene products distributed throughout different categories. The described chromatographic approach provides a significant contribution to the bile protein repertoire and opens new perspectives for the discovery of markers for specific biliary tract diseases.
Journal of Chromatography 01/2008; 1176(1-2):192-205. · 4.53 Impact Factor
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ABSTRACT: The selection of chromatography media and their sequential use represent a major difficulty to isolate a single protein from very crude protein extracts. The process described here consists of two main steps: (i) a rational selection of few media from a relatively large collection and (ii) the definition of the sequence of columns to get the best purity of the target protein. From the first step, one sorbent is selected for its properties to capture the protein to purify, regardless whether other protein impurities are also co-adsorbed; then 5-7 other complementary sorbents are identified to remove impurities but without interacting with the target protein under the same buffering conditions. The second step consists in superimposing sorbents under a cascade manner with the sorbent in charge to capture the target protein located in the last position. Non-adsorbed proteins are eliminated in the flowthrough; other impurities are progressively removed by the sorbent sequence and the target protein is finally desorbed and isolated from the last sorbent using an optimized gradient. All operations are performed with a single adsorption buffer for all columns and all monitoring performed by means of mass spectrometry associated with ProteinChip arrays and polyacrylamide gel electrophoresis. Examples of protein isolation/identification from human serum are described namely thyroxin-binding proteins and transferrin. The first is isolated thanks to a series of dye chromatography media, the second (transferrin) using current chromatographic media. In both cases the target proteins were purified at a level estimated of about 95% and 85%, respectively. Isolated proteins were pure enough for the purpose of formal identification by either peptide fingerprinting or sequencing.
Journal of Chromatography 08/2007; 1156(1-2):188-95. · 4.53 Impact Factor
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ABSTRACT: The complexity of the human serum proteome is attributed to both a large dynamic range of protein abundance, as much as 10 orders of magnitude, and a disproportionate few dozens of proteins representing as much as 99% of the total protein content. These characteristics make it beneficial to use a pre-fractionation step prior to any high-resolution analysis, such as mass spectrometry. The present method describes a unimodal multidimensional chromatography concept to rapidly achieve an effective fractionation of human serum that is directly amenable with surface-enhanced laser desorption/ionization (SELDI)-based mass spectrometry. This method is based on the use of a column composed of a superimposed sequence of sorbents. The assembly is first equilibrated with a single binding buffer and then loaded with the whole crude sample. As the sample crosses the different adsorbent layers proteins within are sequentially trapped according to the complementary properties vis-a-vis of the sorbent. Once the loading and capturing is achieved, the sequence of columns is disassembled and each column, containing different complement of proteins is eluted separately in a single step and under optimal elution conditions. When compared to classical single-chemistry fractionation based on, for example, anion-exchange and pH stepwise elution, the new proposed approach shows much lower protein overlap between fractions, and therefore, greater resolution. This results in a larger number of detectable species, and therefore, reinforces the power of discovery of new biomarkers. A significantly higher sensitivity for low-abundance species was additionally found as evidenced by spiking trials.
Journal of Chromatography 06/2005; 1073(1-2):25-33. · 4.53 Impact Factor
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ABSTRACT: The report describes the use of 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as a ligand for the separation of antibodies by chromatography. The ligand shows a relatively specific adsorption property for antibodies from very crude biologicals at pH 5.0-5.5. At this pH range most of other proteins do not interact with the resin especially when the ionic strength is similar to physiological conditions. Several characterization studies are described such as antibody adsorption in different conditions of ionic strength, pH and temperature. These properties are advantageously used to selectively capture antibodies from very crude feed stocks without dilution or addition of lyotropic salts. Demonstration was made that the adsorption mechanism is neither based on ion exchange nor on hydrophobic associations, but rather as an assembly of a variety of properties of the ligand itself. Binding capacity in the described conditions ranges between 25 and 30 mg/mL of resin. The sorbent does not co-adsorb albumin (Alb) and seems compatible with a large variety of feedstocks. Quantitative antibody desorption occurs when the pH is raised above 8.5. The final purity of the antibody depends on the nature of the feedstock, and can reach levels of purity as high as 98%. Even with very crude biological liquids such as ascites fluids, cell culture supernatants and Chon fraction II + III from human plasma fractionation where the number of protein impurities is particularly large, immunoglobumins G (IgG) were separated at high purity level in a single step.
Journal of Chromatography B 09/2004; 808(1):25-33. · 2.89 Impact Factor
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ABSTRACT: Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed. When components of a protein mixture are deposited upon an array's functionalized surface, an interaction occurs between the array's surface and solubilized proteins, resulting in adsorption of certain species. The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations. In retentate chromatography-mass spectrometry (RC-MS), the "retentate" components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution. Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS). The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention-elution of target proteins and impurity components from ion-exchange surfaces. Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions. The RC-MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures. Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode. Analysis of collected LC fractions showed favorable correlation to results predicted by the RC-MS method.
Journal of Chromatography B 01/2003; 782(1-2):307-16. · 2.89 Impact Factor
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ABSTRACT: A novel chromatography method for the separation of antibodies is described. The adsorption of antibodies on the solid phase involves interaction with a ligand that combines mild hydrophobic characteristics and some degree of molecular recognition with a derivative of pyridine. This combined effect results in the adsorption of antibodies in the absence of lyotropic salts. When environmental pH is changed, the ligand becomes ionically charged, allowing the desorption of antibodies. The mechanism of adsorption, involving hydrophobic associations and ionic related interaction, is here qualified as dual-mode. Studies on the determination of the apparent dissociation constant for immunoglobulins G are presented. Adsorption of antibodies from crude feedstocks typically occurs without adjustment of pH or ionic strength. The sorbent is then washed with a buffer to eliminate protein impurities and, when lowering the environmental pH, antibodies are desorbed. The solid-phase material is used for the separation of antibodies from an ascites fluid and from a cell culture supernatant, followed by a polishing step on an hydroxyapatite column. Preliminary studies, related to the ability of the solid phase to separate antibody fragments, are also reported. In these studies, it has been demonstrated that both Fab and Fc fragments from polyclonal IgG are adsorbed to the solid phase under typical binding conditions. Under other defined physico-chemical conditions (ionic strength and pH), separation of both fragments in a single step has been achieved.
Journal of chromatography. B, Biomedical sciences and applications 06/2001; 755(1-2):37-46.
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ABSTRACT: Efficient harvest and recovery of high-purity monoclonal antibodies was achieved using hydrophobic charge induction chromatography (HCIC). Both simple and complex feedstocks were studied, including protein-free cell culture supernatant and the clarified/concentrated milk of transgenic goats. Viral clearance studies demonstrated a 4-log reduction of MVM virus (minute virus of mice), along with substantial reduction of DNA content. Sorbent characterization studies confirmed that HCIC is based on the pH-dependent behavior of a dual-mode, ionizable ligand. Binding, based on hydrophobic interaction, was achieved under near-physiological conditions, and in the absence of lyotropic salt. Desorption was accomplished under mild conditions--pH 4.0. At this pH, both ligand and antibody carry a net positive charge, and desorption occurs on the basis of electrostatic charge repulsion. pH-based control of chromatographic function was demonstrated. Chromatography on this antibody-selective HCIC sorbent was evaluated as a cost-effective, process-compatible alternative to affinity chromatography protein A sorbents.
Journal of Chromatography 02/2001; 908(1-2):251-63. · 4.53 Impact Factor
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ABSTRACT: Hydrophobic charge induction chromatography is a recently developed method for protein separation based on the use of dual-mode ligands. They are designed in such a way so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligand becomes ionically charged resulting into the desorption of the protein. This method is applied to the separation of antibodies from ascite fluids and culture supernatants from hybridomas cultured in the presence of fetal bovine serum or in protein free environment. Typically adsorption from cell culture supernatants is accomplished without any pH or ionic strength adjustment; the column is then washed with a typical buffer to eliminate protein impurities. Antibodies are then desorbed using acetate buffer, pH 4. Antibody binding capacity is in the range of 30 mg per ml of resin at 10% breakthrough. Antibody purity varies according to the initial feed stock and can reach values higher than 90% in a single pass. One example of antibody purification process involving hydrophobic charge induction chromatography as a capture step followed by a polishing phase with DEAE Ceramic HyperD is described. Longevity and ligand leakage are compatible with large-scale applications.
Bioseparation 02/2000; 9(4):211-21.
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ABSTRACT: New highly dense beaded sorbents suitable for fluidized bed applications of protein separations are presented. They are prepared using porous mineral oxides supporting functional hydrogels responsible for protein interaction. Beads of small diameter (70 microns) are selected to reduce mass transfer resistance. Zirconium oxide was the preferred mineral material due to its high density (5.9 g/ml) allowing high fluidizing liquid velocities (600 cm/h) into columns with a moderate bed expansion (lower than 3). Composite mineral--hydrogel sorbents are evaluated for their ability to rapidly adsorb proteins in fluidized bed and to separate with an appropriate resolution macromolecule mixtures in packed bed. Lysozyme dynamic capacities of 68 and 53 mg per ml of sedimented bed were obtained at fluidizing velocities of 450 and 900 cm/h.
Bioseparation 02/1999; 8(1-5):115-20.
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Journal of Molecular Recognition 02/1998; 11(1-6):107-9. · 3.31 Impact Factor
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ABSTRACT: Soluble chemicals extracted from chromatographic media can contaminate pure biological preparations. These contaminants, which may come from the chemical synthesis of the polymers, could have adverse effects as far as their toxicity is concerned. Ion exchangers made using classical acrylic monomers have been investigated for the presence of traces of monomers which are not converted into polymers. In vitro toxicity investigations have also been performed with the same monomers. The obtained data showed that the amount of free residual monomers was below the sensitivity of the analytical methods (HPLC) for both the main monomers (acidic and alkaline) and the acrylic bifunctional monomer. Toxicity trials showed no adverse effects on human cells in culture. Moreover, no polyploidia induction was evidenced in cells cultured in the presence of monomers.
Journal of Biochemical and Biophysical Methods 05/1996; 32(1):15-25. · 2.33 Impact Factor
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ABSTRACT: Soluble chemicals extracted from chromatographic media can contaminate pure biological preparations. These contaminants that may come from the chemical synthesis of the polymers could have adverse effects as far as their toxicity is concerned. Ion exchangers made using classical acrylic monomers have been investigated for the presence of traces of monomers which are not converted into polymers. In vitro toxicity investigations have also been performed with the same monomers. Obtained data demonstrated that the amount of free residual monomers was below the sensitivity of the analytical methods (HPLC) for both the main monomers (acidic and alkaline) and the acrylic bifunctional monomer. Toxicity trials showed no adverse effects on human cells in culture. Moreover no polyploidia induction was evidenced in cells cultured in the presence of monomers.
Annales Pharmaceutiques Françaises 02/1996; 54(6):251-9.
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ABSTRACT: A new method involving a special sorbent designed to specifically capture virus inactivating solvent-detergent mixtures is described. Its specificity allows the adsorption of these undesirable chemicals with a high capacity so as to treat large amounts of inactivated biological fluids in small-sized columns. Typically, the volume of sorbent that can be used repeatedly is between one-fourth to one-tenth of the sample volume to be treated. Cleaning and sanitization can be done classically while strong oxidizing agents can also be used to sterilize the sorbent.
Annales Pharmaceutiques Françaises 02/1996; 54(3):97-102.
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ABSTRACT: The purification of biomolecules for human therapeutic use by liquid chromatography have been widely extended since 10 years. The preparation of such molecules have to be in accordance with quality requirements and some of them still have to be defined because of their high specificity. As a consequence, each purification step must be validated to guarantee a constant product quality and the absence of any contaminant issued from the solid phases employed. In this context, we developed several techniques allowing the characterization of sorbents and their eventual degradation products in order to determine the level of toxicity of the potential leachables in relation with their mode of use. This procedure could be generalized in the future according to the constraints due to the fractionation process itself.
Annales Pharmaceutiques Françaises 02/1996; 54(3):119-25.
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ABSTRACT: A method involving a sorbent designed to capture specifically virus-inactivating solvent-detergent mixtures is described. Its specificity allows the adsorption of these undesirable chemicals with a high capacity in order to treat large amounts of inactivated biological fluids in small-sized columns. Typically, the volume of sorbent that can be used repeatedly is between one quarter and one tenth of the sample volume to be treated. Cleaning and sanitization can be done by classical methods while strong oxidizing agents can also be used to sterilize the sorbent.
Journal of chromatography. B, Biomedical applications 03/1995; 664(1):119-25.
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ABSTRACT: HyperD ion-exchange media combine the mechanical strength of a rigid polystyrene-mineral composite skeleton with the high protein-binding capacity of a three-dimensional soft gel located inside the skeleton. The skeleton solid matrix is completely filled with functionalized, highly hydrophilic, chemically stable ion-exchange hydrogels. These materials gave very efficient columns for protein separation with superior dynamic capacity, high resolving power and excellent protein recovery. Various protein mixtures were used to study the chromatographic performance of these new stationary phases. Comparisons between different particle size packing materials demonstrated the potential of this ion-exchange material for use on a large scale.
Journal of chromatography. B, Biomedical applications 03/1995; 664(1):225-31.
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ABSTRACT: Some reactive textile dyes have been used for years as biomimetic ligands in protein purification. There has been reluctance, however, to use these dyes on a large scale for therapeutically applicable proteins for fear of possible dye leakage and consequent contamination. Therefore, toxicological data are necessary to quantify the level of this hazard. This study deals with a series of in vitro toxicity investigations with eukaryotic cells (growth, polyploidy, etc.) and with prokaryotic cells (Escherichia coli) for genotoxic studies. Both approaches demonstrated a lack of or slight toxicity for Reactive Blue 2 and Reactive Red 120 and their derivatives over the range 10-62.5 micrograms/ml in several assays.
Journal of chromatography. B, Biomedical applications 03/1995; 664(1):241-6.
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ABSTRACT: Leached ligands from chromatographic packing material submitted to drastic regeneration conditions can contaminate pure biological preparations. These contaminants could have adverse effects from a toxicology point of view that are very poorly documented in liquid chromatography for protein separation. Investigations on toxicity level have been made on released material from immobilized Procion Red HE-3B, after formal identification of the nature of the leached chemical material. Toxicity investigations in vitro involved a number of tests on living cells (eucaryotic and procaryotic) covering different aspects. Behaviour of cells in regular cultures, polyploïdia induction, genotoxicity as well as mechanisms of endocytosis have been studied. Results showed no toxic effects within the range of concentration of dye and dye derivatives studied. Genotoxicity studies in particular did not show any toxic effect over a range of concentration much higher than the regular level of dye leakage from the sorbent.
Journal of Biochemical and Biophysical Methods 01/1995; 29(3-4):269-82. · 2.33 Impact Factor
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ABSTRACT: Toxicity effects related to leached ligands from affinity sorbents that can contaminate biological preparations were investigated in the particular case of immobilized Reactive Blue-2. Initially, identification of the real chemical structure of leached dye has been done by HPLC after incubation in extreme conditions. Toxicity investigations in vitro involving several well known tests showed no toxic effects within the studied range of dye concentration. Cell cultures behaved normally when the adhesion phase was successful; polyploidy induction in human cells by the native dye and its derivatives identified as possible leached material was very similar to standard cultures. Genotoxicity studies did not evidence any toxic effect in E. Coli cultures of dyes themselves or of the same dyes after metabolic activation.
Bioseparation 11/1994; 4(5):299-309.
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ABSTRACT: Some reactive textile dyes are used for years as biomimetic ligands in protein purification. The reluctance to use these performant systems in large scale for therapeutically applicable proteins is related with the possible dye leakage and consequently with problems of contamination. Therefore, toxicology data are necessary to quantify the level of danger in association with sensitive assays. This study deals with a series of in vitro toxicity studies with eucaryotic cells (growth, polyploidia, ...) as well as with procaryotic cells (E. coli) for genotoxic studies. Both approaches demonstrated a total absence of toxicity for all ranges of concentrations investigated for Reactive Blue 2, Reactive Red 120 and their derivatives. Additional experiments done in vivo by the administration of dye solutions to a series of mice confirmed the non toxic character of these dyes in vitro.
Annales Pharmaceutiques Françaises 02/1994; 52(3):137-52.
