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Bernardi S.,Polytechnic of Milan | Getaz D.,ETH Zurich | Forrer N.,Zeochem AG | Morbidelli M.,Polytechnic of Milan | Morbidelli M.,ETH Zurich
Journal of Chromatography A | Year: 2013

Mixed-mode chromatographic materials are more and more often used for the purification of biomolecules, such as peptides and proteins. In many instances they in fact exhibit better selectivity values and therefore improve the purification efficiency compared to classical materials. In this work, a model to describe biomolecules retention in cation-exchange/reversed-phase (CIEX-RP) mixed-mode columns under diluted conditions has been developed. The model accounts for the effect of the salt and organic modifier concentration on the biomolecule Henry coefficient through three parameters: α, β and γ. The α parameter is related to the adsorption strength and ligand density, β represents the number of organic modifier molecules necessary to displace one adsorbed biomolecule and γ represents the number of salt molecules necessary to desorb one biomolecule. The latter parameter is strictly related to the number of charges on the biomolecule surface interacting with the ion-exchange ligands and it is shown experimentally that its value is close to the biomolecule net charge. The model reliability has been validated by a large set of experimental data including retention times of two different peptides (goserelin and insulin) on five columns: a reversed-phase C8 column and four CIEX-RP columns with different percentages of sulfonic groups and various concentration values of the salt and organic modifier. It has been found that the percentage of sulfonic groups on the surface strongly affects the peptides adsorption strength, and in particular, in the cases investigated, a CIEX ligand density around 0.04μmol/m2 leads to optimal retention values. © 2013. Source

Getaz D.,ETH Zurich | Gencoglu M.,ETH Zurich | Forrer N.,Zeochem AG | Morbidelli M.,ETH Zurich
Journal of Chromatography A | Year: 2010

Caustic regeneration procedures are often used in chromatographic purification processes of peptides and proteins to remove irreversibly bound impurities from the stationary phase. Silica-based materials are the most commonly used materials in reversed phase chromatography of peptides. Their limited chemical stability at high pH can be, however, problematic when high pH column regeneration (i.e. cleaning in place) is required. The effect of cleaning in place on the surface chemistry of the stationary phase has been investigated using the Tanaka test. It has been shown that the high pH treatment does not significantly affect the hydrophobicity of the material, but it strongly increases its silanol activity. A representative peptide purification process has been used to investigate the impact of cleaning in place on the separation performance. It has been shown that the caustic regeneration increases the peptide retention at high pH (pH 6.5), due to the interactions between the peptide and the negatively charged silanol groups. These unwanted interactions reduce the separation performances by decreasing the selectivity between the late eluting impurities and the main peptide. However, it has been shown that the effect of the silanol groups on the peptide adsorption and on the separation performance can be minimized by carrying out the purification process at low pH (pH ∼ 2). In this case, the silanol groups are protonated and their electrostatic interactions with the positively charged analyte (i.e. peptides) are suppressed. In these conditions, the peptide adsorption and the impurity selectivity is not changing upon high pH column regeneration and the separation performance is not affected. © 2010 Elsevier B.V. Source

Getaz D.,ETH Zurich | Dogan N.,ETH Zurich | Forrer N.,Zeochem AG | Morbidelli M.,ETH Zurich | Morbidelli M.,Polytechnic of Milan
Journal of Chromatography A | Year: 2011

The influence of the pore size of a chromatographic reversed phase material on the adsorption equilibria and diffusion of two industrially relevant peptides (i.e. a small synthetic peptide and insulin) has been studied using seven different reversed phase HPLC materials having pore sizes ranging from 90. Å to 300. Å The stationary phase pore size distribution was obtained by inverse size exclusion measurement (iSEC). The effect of the pore size on the mass transfer properties of the materials was evaluated from Van Deemter experiments. It has been shown that the lumped mass transfer coefficient increases linearly with the average pore size. The Henry coefficient and the impurity selectivity were determined in diluted conditions. The saturation capacity of the main peptides was determined in overloaded conditions using the inverse method (i.e. peak fitting). It was shown that the adsorption equilibria of the peptides on the seven materials is well described by a surface-specific adsorption isotherm. Based on this a lumped kinetic model has been developed to model the elution profile of the two peptides in overloaded conditions and to simulate the purification of the peptide from its crude mixture. It has been found that the separation of insulin from its main impurity (i.e. desamido-insulin) was not affected by the pore size. On the other hand, in the case of the synthetic peptide, it was found that the adsorption of the most significant impurity decreases with the pore size. This decrease is probably due to an increase in silanol activity with decreasing pore size. © 2011 Elsevier B.V. Source

Zeochem Ag | Date: 2013-03-21

A material for reverse phase chromatography comprises surface modifying apolar and charged groups bound to a solid support, said charged groups being present in amounts of about 0.25 to about 22% of the surface modifying groups, or in amounts of about 0.01 mol/m

Khalaf R.,ETH Zurich | Forrer N.,Zeochem AG | Buffolino G.,Zeochem AG | Butte A.,ETH Zurich | Morbidelli M.,ETH Zurich
Journal of Chromatography A | Year: 2015

Reversed-phase (RP) chromatography is one of the main tools for the preparative purification of therapeutic peptides. In previous works [1,2], a new type of RP chromatography, doped reversed-phase chromatography (DRP) was presented. By adding small amounts (up to 15% of the surface ligands) of repulsive ion exchange ligands to a traditional RP material, significant improvements in peptide purification performance were observed, at the same or in similar operating conditions. These improvements included increases in selectivity in diluted conditions (up to twice as high), increases in yield in preparative conditions (up to 20% higher) and in productivity in preparative conditions (up to twice as high), when compared to RP materials [2]. A proper physical model is developed in this work to quantitatively explain and rationalize this behavior. The developed model is then used to correctly fit the retention data of several peptides in different buffering conditions. The increase in selectivity is related to a controlled decrease in free surface area available for adsorption due to the ionic ligands creating a repulsive sphere the analytes cannot enter. This decrease in adsorption surface is calculated using Debye-Hückel theory, and in combination with linear solvent strength theory, allows for the quantitative description of peptide retention on DRP media. © 2015 Elsevier B.V. Source

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