Pierre Girot

Composite affinity sorbents and their cleaning in place

Making large-scale affinity sorbents that are reusable under acceptable hygienic conditions implies specific treatments for cleaning in place with known aqueous solutions of chemical agents. However, common agents such as sodium hydroxide are frequently considered too drastic for the stability of macromolecular biologically active immobilized ligands. According to a large series of trials, it was found that only a mixture of sodium hydroxide and ethanol was actually effective in sterilizing a sorbent in a single step. When hydroxide or an ethanol-acetic acid mixture were used alone, they were not totally efficient in the inactivation of sporulated Bacillus subtilis. Conversely, they were efficient when used sequentially. All these solutions were able to remove pyrogens from chromatographic sorbents. As the sterilizing solutions contained a certain amount of ethanol, the most suitable chromatographic affinity sorbents had to be based on an incompressible matrix. When washing an affinity silica sorbent that had proteins as ligands with solutions such as sodium hydroxide, ethanol-acetic acid or ethanol-sodium hydroxide, it was found that certain sorbents were able to tolerate the treatments without a noticeable decrease in their biochemical activity.

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.

Characterization of very dense mineral oxide–gel composites for fluidized-bed adsorption of biomolecules

Efficient design of fluidized-bed biomolecule adsorption from crude feed stock requires particles with elevated density, large adsorption capacity and broad chemical stability. Moreover, combinations of small particle diameters with high densities allow for high fluidization velocities while preserving a rapid mass transfer.This approach has been implemented by combining stable porous mineral oxide of high density (2.2, 4.7, 5.7, 9.4 g/ml) with functionalized hydrogels. The cross-linked hydrogel derivative fills the internal porosity of the beads and provides a high equilibrium binding capacity.Various porous mineral oxides (silica, titania, zirconia and hafnia) have been characterized in term of fluidization behavior, surface reactivity and chemical resistance to harsh CIP procedures. Porous zirconia particles were also modified into ion-exchangers by suitable surface modification and intraparticle polymerization of functionalized stable derivatives of acrylic monomers. Back-mixings in fluidized bed columns were analyzed by residence time distribution analysis of inert tracers. 328 and 218 mixing plates per meter were found for respectively, bed expansions of 1.7 and 2.9. The dynamic protein adsorption behaviors of zirconia-based polymeric anion-exchange sorbents were obtained in fluidized-bed, using BSA as model protein. A dynamic binding capacity of 62 mg/ml was observed at a fluidizing velocity of 320 cm/h. These investigations substantiate the favorable physical and chemical characteristics anticipated for dense composite beads for use as fluidized bed adsorbents.

Silica—dextran sorbent composites and their cleaning in place

Abstract Large-scale liquid chromatography requires sorbents that are mechanically stable in order to ensure repeated cycles without shrinkage. The industrial separation of biological materials moreover implies working in apyrogenic and sterile conditions. Dextran-coated silica used as a chromatographic sorbent was treated with different sanitizing solutions after bacterial contamination. It was demonstrated that most of the strains used were inactivated by using alkaline solutions or ethanolic acetic acid. However, the sporulated form of Bacillus subtilis , which seemed more resistant than other strains, was inactivated only when alkaline solutions contained more than 40% ethanol. All these treatments were also very effective for pyrogen removal and did not indicate significant degradation of the silica moiety or the chromatographic performance of the sorbent.

A pI-based protein fractionation method using solid-state buffers

The analysis of very complex proteomes is dependent on efficient fractionation methods with low level of carry over from fraction to fraction. Among various possibilities the separation by ranges of isoelectric points for further analysis appears as attractive, but current methods involving an electrically driven migration in the presence of ampholyte carriers are not exempt of technical complications. In the present work a new separation concept is described involving the use of so-called solid-state buffers, in association with ion exchangers, to separate protein categories of different pI ranges with a low level of protein overlapping. Resin blends packed in separated columns are used under a cascade configuration of increasing or decreasing pH and, once proteins of different pI are adsorbed by individual resin blends, the columns are dissociated. From each column protein mixtures corresponding to a given pI range are collected by competitive desorption with salts so as to be ready for proteomic analysis. The process is rapid and does not involve electrical fields nor addition of carrier ampholyte material. The presence of potassium chloride during the separation prevents protein precipitation at the vicinity of their isoelectric points. The fractions thus obtained can be used for two dimensional electrophoresis and mass spectrometry analysis after the removal of salts.

Immobilized cibacron blue--leachables, support stability and toxicity on cultured cells.

Although immobilized dyes are widely used on the laboratory scale and have good potential for industrial applications, they are still subject to some reservations. Little information is available about dye leakage and toxicity, which seriously hinders the use of such supports in the production of pure proteins. Investigations of the leakage mechanism and the in vitro toxicity of the native dye and of that leached from the column are reported. The possible presence of traces of dye in the purified biological materials necessitates the availability of sensitive analytical tests. The preparation and preliminary isolation of dye antibodies as a first step in the development of an immunohistochemical assay of leached dyes are also described.

New method for the separation of oligonucleotides by chromatography on acriflavin—agarose

Iu the fields of molecular genetics and nucleic acid research, the synthesis and preparation of homoand hetero-oligonucleotides are of growing interest. Generally, oligonucleotide separation is performed by ion-exchange chromatography [l-5] or by high-performance liquid chromatography (HPLC) (review [6]). Separation based on ion exchange allows for limited resolution since the charge difference between successive oligomers is small. However, HPLC requires expansive apparatus. Such drawbacks led to the synthesis of new adsorbents for oligonucleotides and related compounds separation. Separation of aromatic and heterocyclic compounds may be performed by charge-transfer chromatography employing solid sup ports to which acriflavin has been covalently bound [7,8]. Such charge-transfer gels were used in [9] to separate 5’-AMP from cyclic 3’,5’-AMP. The separation of single from double-stranded DNA can also be achieved through the use of acriflavin-agarose gels [lo]. Adsorbents consisting of similar dyes covalently bound to polyacrylamide gels have been used for basepair-specific fractionation of sheared DNA [ 1 l] or for structure-specific separation of supercoiled DNA from released or linear forms [ 11,121. Such separations are based on the effects of electron-charge transfer, hydrogen bonding and electrostatic interaction in promoting the formation of intercalating dye complexes. 5’-TMP was purchased from Boehringer (France). Oligo(dT) was synthesized from the pyridinium salt of 5’-TMP under anhydrous conditions and in the presence of dicyclohexylcarbodiimide (AldrichEurope, Belgium) according to [ 13].Oligo(rI) was a gift from Professor M. Michelson (Institut Curie, Paris). Hetero-oligonucleotides were synthesized using Escherichia coli polynucleotide phosphorylase as in [14,15]. Essentially, the reaction mixture contained 100 mM Tris-HCl buffer (pH 8.5), 0.3 M NaCl, 10 mM 2-mercaptoethanol, 10 mM MnClz oligo(dT), (Collab. Res. Mass.) as primer 5’-dNDP (dNDP/primer = 1:5) and 5 units polynucleotide phosphorylase/ml. The mixture was incubated at 37’C for several hours and stopped by the addition of 50 mM EDTA. Acriflavin-ultrogel A4R type I was obtained from Reactifs IBF (France) or prepared by a modification of the original method in [7] using 4% agarose gel beads instead of dextran gel. DEAE-Trisacryl M was obtained from Reactifs IBF. HPLC chromatography was performed with a RPC5 column [6] consisting of a polychlorotrifluoroethylene support coated with methyl-trialkyl-ammonium chloride. All other chemical agents were of analytical grade.

Immunoenzymatic assay of dyes currently used in affinity chromatography for protein purification.

Cibacron Blue F3-GA, Basilen Blue E3-G and Procion Red HE-3B are dyes currently used in affinity purification, and are commonly determined by spectrophotometry with limited sensitivity. An assay method is described based on a specific immunochemical recognition of the dyes amplified by a final enzymatic reaction. The sensitivity is close to 1 ng/ml of dye and the method is applicable any time that sensitive and accurate results are necessary. This method has actually been applied with success to the determination of trace amounts of dyes in the presence of affinant protein. The method was also applied to the demonstration of dye leaching from affinity sorbents when treated under acidic and/or alkaline conditions.