An isoelectrically trapped enzyme reactor operating in an electric field

Righetti, Pier Giorgio; Bossi, Alessandra Maria

doi:10.1002/elps.1150190705

Cited by 23 publications

(19 citation statements)

References 30 publications

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“…This technique is a powerful tool for protein separation, and has been used widely in biochemistry, life sciences and clinical studies [41,42,43,44,45,46,47,48]. Nearly 30 000 papers have been published in this area, and the number is still increasing rapidly.…”

Section: Isoelectric Focusingmentioning

confidence: 99%

Analytical equilibrium gradient methods

Wang

Tolley

LeFebre³

et al. 2002

Anal Bioanal Chem

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Analytical equilibrium gradient methods are non-linear separation methods in which the separation mechanism involves a force gradient along the separation channel. These methods can be classified into two categories: those in which the gradient is a field gradient applied along the separation channel (i.e., field gradient), and those in which the channel is subjected to a constant field with a gradient formed in some other property (i.e., constant field). Standard deviation of peak width, resolution and peak capacity are important parameters in characterizing equilibrium gradient methods, and general expressions can be obtained from considering both the point of force acting on the analyte and the basic flux equation. Several successful examples, such as density gradient sedimentation, isoelectric focusing and electromobility focusing are discussed. Based on equilibrium gradient methods in the field gradient category, a method to dynamically improve peak capacity is described. An example of such an approach is given using electromobility focusing.

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Section: Isoelectric Focusingmentioning

confidence: 99%

Analytical equilibrium gradient methods

Wang

Tolley

LeFebre³

et al. 2002

Anal Bioanal Chem

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“…Indeed, the poor solubility and hydrophobic interactions, responsible for wall adsorption, can lead to protein precipitation, mostly in a pH region close to their pI. For this reason, some additives are often used to improve the solubility of hydrophobic proteins, such as mixtures of thiourea, urea and a surfactant [11,12]. The presence of surfactants, however, is not suitable for CE-MS coupling, a final step that cannot be spared in proteomic analysis [13].…”

Section: Introductionmentioning

confidence: 98%

Evaluation of capillary isoelectric focusing in glycerol‐water media with a view to hydrophobic protein applications

Busnel¹,

Varenne²,

Descroix³

et al. 2005

Electrophoresis

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Capillary isoelectric focusing (CIEF) separations are usually performed with neutral coated fused-silica capillaries in aqueous anticonvective media. Glycerol, a very viscous solvent (eta = 945 mPa x s at 25 degrees C), known to help stabilize any kind of proteins and solubilize hydrophobic ones, was tested as an alternative to using commercial gels. Viscosity and electroosmotic mobility were measured as a function of gel or glycerol content in water, and a 30:70 v/v glycerol-water medium appeared as a good compromise for performing CIEF in a bare fused-silica capillary without imposing too high a viscosity. To demonstrate the feasibility of this new CIEF system, a standard mixture of nine model proteins was separated according to their pI with a good agreement between experimental and literature aqueous pIs. Moreover, better resolution was achieved with this system than with the conventional aqueous CIEF system, as two of the model proteins could not be separated in the latter system. Glycerol-water CIEF in bare silica capillary was next applied to the separation of horse radish peroxidase, a complex mixture of protein isoforms. The good concordance with the separation obtained by the conventional CIEF system indicated the adequacy of this new system. Finally, as anticipated from the results obtained for the separation of bacteriorhodopsin, a membrane protein, glycerol-water CIEF performed in bare silica capillary appears to be a promising alternative to conventional aqueous CIEF for hydrophobic protein characterization, under their native form.

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“…This could be explained by a selective ion pairing between citrate and glycoforms [58][59] that increased differences in the charge-to-hydrodynamic radius ratio of glycoforms [60]. Acidic isoelectric buffers as described by Righetti's group [61][62][63][64][65] and reviewed extensively [66,67] were then investigated. The major advantage of these buffers is that their very low conductivity allows the use of high concentrations and high voltage without exacerbating Joule heating effects, thus reducing separation time.…”

Section: Optimization Of the Separation By Czementioning

confidence: 99%

CZE for glycoform profiling and quality assessment of recombinant human interleukin‐7

Alahmad

Tran

Duboeuf³

et al. 2009

Electrophoresis

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The aim of the present work was to develop a simple high-resolution CZE method for glycoform profiling and quality assessment of recombinant human interleukin-7 (rhIL-7) produced in Chinese hamster ovary cells. Classical and isoelectric buffers and several monoamines used as alkaline component of BGE at very low concentration have been investigated in order to optimize the resolution. The best separation was achieved using a fused-silica capillary and a buffer composed of 25 mM citrate/triethanolamine at pH 2.6. Under these acidic conditions, triethanolamine was able to reverse EOF favorably and to limit rhIL-7 adsorption. This method allowed the separation of four groups of peaks ranging from low to high-sialylated glycoforms. An extensive study on inter-run rinsing procedures has been conducted. Rinsing with 50 mM SDS was retained to achieve the optimal repeatability. Excellent intermediate precision was obtained for migration time (RSD < 0.6%), while RSD for intraday studies were only less than 2.9%. Satisfactory inter and intraday repeatabilities were also observed for relative peak area. We finally demonstrated that reliable information could be obtained to address comparability studies and demonstrate batch-to-batch consistency of biomanufactured rhIL-7.

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An isoelectrically trapped enzyme reactor operating in an electric field

Cited by 23 publications

References 30 publications

Analytical equilibrium gradient methods

Analytical equilibrium gradient methods

Evaluation of capillary isoelectric focusing in glycerol‐water media with a view to hydrophobic protein applications

CZE for glycoform profiling and quality assessment of recombinant human interleukin‐7

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