Control of Raw Pork Liver Sausage Production Can Reduce the Prevalence of HEV Infection.

After an acute hepatitis E (HEV) outbreak in Southern Switzerland, in January 2017 the local public health authorities started an active program of food chain control and public education. In this retrospective study, we analysed all laboratory-confirmed acute cases of HEV infection diagnosed between 2014 and 2020. In the period before the public health intervention, the number of cases increased steadily from 2014 (4 of 40 tests, 10%) reaching a peak in the last quarter of 2016 (42 of 285 tests, 14.7 %). Afterwards, the number of positive cases decreased steadily, reaching its lowest value (0.3%) in the second quarter of 2019. There was a statistically significant difference between the frequency of positive cases and period of testing, i.e., before and after the introduction of the public health interventions. Our study shows that active public health measures to control sausages containing raw pork liver can reduce the prevalence of HEV infection.

Model-based description of peptide retention on doped reversed-phase media.

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.

Doping reversed-phase media for improved peptide purification.

The purification of therapeutic peptides is most often performed using one or more reversed phase chromatography steps. This ensures high purities while keeping the costs of purification under control. In this paper, a doped reversed phase chromatographic material is tested and compared to traditional reversed phase materials. The doping consists of adding limited amounts of ion exchange ligands to the surface of the material to achieve orthogonal separation and increase the non-hydrophobic interactions with the surface. These ionic groups can either be attractive (opposite charge), or repulsive (same charge) to the peptide. The benefit of this new doped reversed phase material is shown through increases in selectivity in diluted conditions and yield and productivity in overloaded (i.e. industrial) conditions. It is the conjectured that all performance characteristics should increase using repulsive doping groups, whereas these characteristics should decrease when using attractive doping groups. This conjecture is shown to be true through several examples, including purifications of industrially relevant peptide crudes, in industrially relevant conditions. Moreover, the effect of ionic strength and organic modifier concentration was explored and shown to be in line with the expected behavior.

Modeling of mixed-mode chromatography of peptides.

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, ß and γ. The α parameter is related to the adsorption strength and ligand density, ß 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/m(2) leads to optimal retention values.

Influence of the pore size of reversed phase materials on peptide purification processes.

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.

Effect of high pH column regeneration on the separation performances in reversed phase chromatography of peptides.

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 approximately 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.

Role of the ligand density in cation exchange materials for the purification of proteins.

The performance of functionalized materials, such as cation exchange resins, is dependent not only on the ligand type and ligand density, but also on the pore accessibility of the target molecule. In the case of large molecules such as antibodies this latter parameter becomes crucial, because the size of such molecules falls somewhere inside the pore size distribution of the resin. The influence of the ligand density and accessibility on the overall performance of the material is explored systematically. Five different materials, having the same chemistry as the strong cation exchange resin Fractogel EMD SO(3)(-) (M) , have been analyzed. These materials only differ in the ligand density. It is shown that the ligand density directly influences the porosity of the materials as well as the pore diffusivity and the dynamic binding capacity. For a given purification problem an optimal ligand density can be found. Based on the above results a new material is proposed, showing superior properties in terms of dynamic binding capacity. This is achieved by an optimization of the ligand density and by a decrease of the particle size of the stationary phase. The material properties are modeled with a general rate model. Further simulations were conducted to evaluate the performance of the new material in comparison with a conventional resin.

Chromatographic behavior of a polyclonal antibody mixture on a strong cation exchanger column. Part I: Adsorption characterization.

In this work, the adsorption of a polyclonal antibody (IgG) on a preparative strong cation exchanger is described in detail. In a first stage, the polyclonal mixture is characterized using both chromatographic and non-chromatography methods. This analysis suggested that the mixture can be simplified by lumping the different components of the IgG into two "macro-components", referred to as pseudo-variants in the following. An analytical method for the determination of the concentrations of the two pseudo-variants is developed. Based on this, the mass transport and the adsorption isotherm parameters are determined experimentally using only well known sort-cut methods. This analysis is evidencing the complexity of the characterization when operating in the presence of slow diffusing mixtures of components. Even in the presence of strong competition, mass transport plays a major role in determining the peak shape. In frontal analysis, competition is still operative after full saturation of the column.

Chromatographic behavior of a polyclonal antibody mixture on a strong cation exchanger column. Part II: Adsorption modelling.

The adsorption of a polyclonal IgG mixture on a strong cation exchanger column is characterized using a detailed multi-component pore model. This model is explicit in all transport parameters and includes salt dependent isotherms. As discussed in the first part of this work, the IgG mixture can be simplified by considering two pseudo-variants only. Linear gradient experiments are used to fit the salt dependent adsorption isotherms and the mass transport parameters for the two pseudo-variants. Using the model, breakthrough curves are predicted with good accuracy. The model is also implemented to visualize the axial and radial concentration profiles of the two pseudo-variants in the column during a loading experiment.