Contribution of solid-phase hexapeptide ligand libraries to the repertoire of human bile proteins.

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.

A new general approach to purify proteins from complex mixtures.

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.

A simplified monobuffer multidimensional chromatography for high-throughput proteome fractionation.

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.

A dual-mode approach to the selective separation of antibodies and their fragments.

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.

Comparison of hydrophobic charge induction chromatography with affinity chromatography on protein A for harvest and purification of antibodies.

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.

New method for the selective capture of antibodies under physiological conditions.

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.

Quantification and in vitro toxicity studies of extractable chemicals from synthetic ion exchangers.

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.

[Chromatographic support to remove solvent/detergent mixture utilized as viral attenuation agent]. / Supports chromatographiques pour éliminer les mélanges solvant/détergent employés comme agents d'atténuation virale.

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.

[Quantification and studies of in vitro toxicity of chemicals extracted from synthetic ion exchange media]. / Quantification et études de toxicité in vitro des extractibles d'échangeurs d'ions synthétiques.

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.

Specific sorbent to remove solvent-detergent mixtures from virus-inactivated biological fluids.

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.

Preparative high-performance liquid chromatographic separation of proteins with HyperD ion-exchange supports.

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.

Synthesis and separation of optically active compounds. Part I.

Biological processes involve a high degree of stereoselectivity. Considering that pharmacological activity is very often associated with only one enantiomer, regulation authorities have defined new guidelines in requiring chiral drugs to be marketed as pure individually safe and effective enantiomers. If large scale production of pure stereoisomers has met until recently a great difficulty to become economically acceptable, the current state of the art in asymmetric synthesis and chiral separation, provides however many industrial applications. Among the methods currently used, asymmetric synthesis, using chiral auxiliaries or chiral catalysts such as enzymes or metal complexes, allows to create new asymmetric centers, starting from prochiral molecules. Regarding racemate resolution, asymmetric synthesis offers the real economical advantage to produce exclusively the targeted enantiomer. However, resolution methods allowing access to both enantiomers, are very useful at preparative scale for new chiral drugs when preclinical studies are requested for each enantiomer. Large scale racemate resolution can be achieved by crystallization, enzymatic resolution and even more recently by liquid chromatography. Although the "unwanted" enantiomer is also produced, the feasibility of its racemization for recycling has made resolution methods widely used in industry. It is clear today that in most cases, pharmacologically active molecules having an asymmetric center will have to be marketed as optically pure drugs.

Synthesis and separation of optically active compounds. Part II.

This review is the second part of an article devoted to the synthesis and separation of optically active compounds. This article deals mainly with chiral chromatography which represents an important component of the state of art for the preparation of enantiomers, as illustrated by a description and a classification of the main packing materials available to date. The various phases are based on different principles of molecular recognition and show advantages and drawbacks according to the molecules to separate. The preparative aspect of this method is particularly emphasized here and in the same context, trends and future developments are mentioned.

[Chromatographic sorbents for the preparative separation of proteins]. / Supports chromatographiques pour la séparation préparative des protéines.

Chromatography separation steps are technologically very well accepted in downstream bioprocessing, but progresses are expected on productivity level as well as on cleanability. At large production scale, the driving force for purification processes is the throughput intended as the amount of material separated at a given velocity and for a defined level of purity. The possibility to play on some phases of the separation cycle and on the variation of dynamic binding capacity are clearly useful to diminish the separation time while maintaining high levels of loads. On the other hand, to sanitize properly the sorbents, it has been found that only very strong solutions such as alkaline ethanol or alternated acidic-alkaline solutions are effective. In this general situation, special sorbents are now proposed, withstanding stringent operational situations.