196,825 research outputs found
Exploring the potential of zwitterionic teicoplanin-based CSPs by using macroporous superficially porous silica particles 2.7 μm 160Å and 3.4 μm 400Å
No full textDuring the last years, the research in the enantioselective Ultra High Performance Chromatography (eUHPC) is going to push the limits of high efficient and ultrafast analyses. In this work, novel Chiral Stationary Phases (CSPs) were prepared by covalently bonding the teicoplanin selector (TE_A2-2) on Halo 2.7μm 160Å and 3.4μm 400Å Superficially Porous silica Particles (SPP). An innovative bonding protocol allowed to obtain a zwitterionic teicoplanin based CSP, which was used to produce the already known UHPC-FPP-Titan-Tzwitt CSP based on 1.9 μm 120Å Fully Porous monodispersed silica Particles (FPP) and UHPC-SPP-Halo90-Tzwitt CSP 2.0 μm [1-3]. Columns with an internal diameter of 4.6 mm and different lengths (50 and 100 mm) were packed with all CSPs and characterized in terms of permeability, efficiency and thermodynamic under HILIC conditions. The kinetic performance was evaluated through the use of van Deemter curves. The UHPC-SPP-Halo160-Tzwitt 2.7 μm column exhibited extremely high efficiencies on both achiral (>323,000 theoretical plates/meter, N/m; hr: 1.14) and chiral compounds (>240,000 N/m; hr: 1.53) under HILIC conditions, attesting the high potential of this CSP from the kinetic viewpoint. An extreme efficiency was recorded by using the HPC-SPP-Halo400-Tzwitt 3.4 μm column which allowed to achieve an efficiency of 280’000 N/m on naphthalene (hr: 1.05). Furthermore, taking into account the thermodynamic viewpoint, the UHPC-SPP-Halo160-Tzwitt 2.7 μm exhibited the highest resolution power (Rs/tr,2) thanks to its enantioselectivity values because of the higher selector density on the silica matrix. In conclusion, in this study we demonstrate the potential of the use of SPP macroporous silica particles in the UHPLC chiral field opening an interesting scenario in the chiral chromatography area.
[1] O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, J. Chromatogr. A, 1427 (2016) 55–68.
[2] O.H. Ismail, M. Antonelli, A. Ciogli, C. Villani, A. Cavazzini, M. Catani, F. Gasparrini, J. Chromatogr. A, 1520 (2017) 91–102
[3] O.H. Ismail, M. Antonelli, A. Ciogli, M. De Martino, M. Catani, C. Villani, A. Cavazzini, M. Ye, D.S. Bell, F. Gasparrini, J. Chromatogr. A., 1576 (2018), 42-50
Process intensification of the polishing step of a bioactive peptide through Multicolumn Countercurrent Solvent Gradient Purification
No full textPharmaceutical products, including peptides, must satisfy very strict purity specifications, because of quality and safety reasons. Therefore, the necessity to operate one or more purification steps to obtain high quality drugs is indisputable. Critical impurities chemically very similar to the target product are generated during the synthesis and are generally removed by means of preparative single-column chromatographic techniques (=batch methods) [1,2]. Batch methods struggle to separate completely the peptide of interest from other groups of impurities, because of their similarity and of high loading of sample processed in preparative conditions, which cause peaks overlapping [3]. The typical situation encountered in these cases is the so-called center-cut separation, where the target elutes as intermediate between two other groups of impurities less and more retained respectively. The direct consequence of this apparently insurmountable overlapping is a yield-purity trade-off, a limit intrinsic to batch chromatography according to which it is possible to obtain either high purity or high recovery of the peptide of interest, depending on whether the overlapping windows are collected or not [4]. This trade-off leads to drawbacks in the overall economy of the process. Multicolumn chromatographic processes, operating in continuous and countercurrent mode, can alleviate this limitation by performing internal recycling of the overlapping portions of the chromatogram [5]. The technique used in the frame of this research is twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which has been applied to the purification of an industrial crude of a bioactive decapeptide. It has been demonstrated that MCSGP leads to promising results, including a remarkable improvement in process performance (up to 6 times higher) from the point of view of recovery, productivity and solvent consumption, with respect to the corresponding batch run. The automation of the process on industrial scale would lead to great reproducibility which would reflect in improved consistency in product quality.
References
[1] De Luca, C.; Felletti, S.; Lievore, G.; Buratti, A.; Vogg, S.; Morbidelli, M.; Cavazzini, A.; Catani, M.; Macis, M.; Ricci, A.; Cabri, W. J Chromatogr A 2020, 1625, 1-7.
[2] De Luca, C.; Felletti, S.; Lievore, G.; Chenet, T.; Morbidelli, M.; Sponchioni, M.; Cavazzini, A.; Catani, M. Trends Analyt Chem 2020, 132, 1-8.
[3] Vogg, S.; Ulmer, N.; Souquet, J.; Broly, H.; Morbidelli, M. Biotechnol J 2019, 1800732, 1-8.
[4] Müller-Späth, T.; Ströhlein, G.; Lyngberg, O.; Maclean, D. Chem Today 2013, 31, 56-60.
[5] Steinebach, F.; Müller-Späth, T.; Morbidelli, M. Biotechnol J 2016, 11, 1126-1141
Multicolumn Countercurrent Solvent Gradient Purification (MCSGP) process for the intensification of the polishing step of a bioactive peptide mixture
No full textPharmaceutical products, including peptides, must satisfy very strict purity specifications, because
of quality and safety reasons. Therefore, the necessity to operate one or more purification steps to
obtain high quality drugs is indisputable. Critical impurities chemically very similar to the target
product are generated during the synthesis and are generally removed by means of preparative
single-column chromatographic techniques (=batch methods) [1,2]. Batch methods struggle to
separate completely the peptide of interest from other groups of impurities, because of their
similarity and of high loading of sample processed in preparative conditions, which cause peaks
overlapping [3]. The typical situation encountered in these cases is the so-called center-cut
separation, where the target elutes as intermediate between two other groups of impurities less and
more retained respectively. The direct consequence of this apparently insurmountable overlapping
is a yield-purity trade-off, a limit intrinsic to batch chromatography according to which it is possible
to obtain either high purity or high recovery of the peptide of interest, depending on whether the
overlapping windows are collected or not [4]. This trade-off leads to drawbacks in the overall
economy of the process. Multicolumn chromatographic processes, operating in continuous and
countercurrent mode, can alleviate this limitation by performing internal recycling of the
overlapping portions of the chromatogram [5]. The technique used in the frame of this research is
twin-column Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which has been
applied to the purification of an industrial crude of a bioactive decapeptide. It has been
demonstrated that MCSGP leads to promising results, including a remarkable improvement in
process performance (up to 6 times higher) from the point of view of recovery, productivity and
solvent consumption, with respect to the corresponding batch run. The automation of the process on
industrial scale would lead to great reproducibility which would reflect in improved consistency in
product quality.
[1] C. De Luca; S. Felletti; G. Lievore; A. Buratti; S. Vogg; M. Morbidelli; A. Cavazzini; M.
Catani; M. Macis; A. Ricci; W. Cabri, J Chromatogr A 2020, 1625, 1-7.
[2] C. De Luca; S. Felletti; G. Lievore; T. Chenet; M. Morbidelli; M. Sponchioni; A. Cavazzini; M.
Catani, Trends Analyt Chem 2020, 132, 1-8.
[3] S. Vogg; N. Ulmer; J. Souquet; H. Broly; M. Morbidelli, Biotechnol J 2019, 1800732, 1-8.
[4] T. Müller-Späth; G. Ströhlein; O. Lyngberg; D. Maclean, Chem Today 2013, 31, 56-60.
[5] F. Steinebach; T. Müller-Späth; M. Morbidelli, Biotechnol J 2016, 11, 1126-1141
The complex process of cellular vitamin A uptake: first insights by NMR and other biophysical techniques
No full textVitamin A is essential for diverse aspects of life, ranging from embryogenesis to the proper functioning of most adult organs. It circulates in blood bound to serum retinol binding protein (RBP) and is transported into cells by a membrane receptor termed Stimulated by Retinoic Acid 6 (STRA6) [1]. The mechanism of the STRA6-mediated translocation of retinol from holo-RBP into target cells appears to be unique [2]. There are also evidences that a specific binding site for the intracellular carriers (CRBPs) might exist on the cytoplasmic side of the membrane [3] and that CRBP-I plays a key role other than simply to sustain retinol transport [4]. However, until now it is not clear whether the apo-CRBPs may bind STRA6 or they interact with the membrane for retinol uptake.
To gain first insights into this complex process, we have investigated the interactions of CRBP-I and CRBP-II with biomembrane mimetic systems. NMR experiments were performed at different protein:vesicles molar ratios and by varying the composition of the phospholipid liposomes and the ionic strength. Chemical shifts perturbations and line shape analysis provided insights into the interacting residues and proteins conformational dynamics. As the signals were broadened beyond detection at latest steps of the titration, the NMR data have been complemented by other biophysical measurements.
The results revealed striking differences between CRBP-I and CRBP-II, despite they exhibit the same fold (a beta-barrel with two short alpha-helices) and identical retinol-binding motifs. Moreover, the interactions of the two homologs with the lipid bilayers depend upon the phospholipid composition and ionic strength.
These new evidences complement the lessons learned from our former studies which had suggested that the two primary cellular retinol carriers exhibit different mechanisms of ligand uptake [5, 6]. These differences may account for their distinct functional roles in the modulation of intracellular retinoid metabolism.
References
[1] R. Kawaguchi, J. Yu, J. Honda, J. Hu, J. Whitelegge, P. Ping, P. Wiita, D. Bok, and H. Sun Science 315, 820-825 (2007).
[2] R. Kawaguchi, J. Yu, M. Ter-Stepanian, M. Zhong, G. Cheng, Q. Yuan, M. Jin, G.H. Travis, D. Ong, and H. Sun ACS Chemical Biology 6, 1041-1051 (2011).
[3] C. Redondo, M. Vouropoulou, J. Evans, and J.B.C. Findlay The FASEB J. 22, 1043-1054 (2008).
[4] D.C. Berry, S.M. O'Byrne, A.C. Vreeland, W.S. Blaner, and N. Noy Mol. Cell. Biol. (2012) in press.
[5] T. Mittag, L. Franzoni, D. Cavazzini, B. Schaffhausen, G.L. Rossi, and U.L. Günther J. Am. Chem. Soc. 128, 9844-9848 (2006).
[6] L. Franzoni, D. Cavazzini, G.L. Rossi, and C. Lücke J. Lipid Res., 51, 1332-1343 (2010)
STUDY OF THE ADSORPTION EQUILIBRIA OF A PHARMACEUTICAL RELEVANT PEPTIDE IN RP-LC THROUGH ADVANCED NUMERICAL MEANS
No full textDue to their biologic activity, many peptides are used in cosmetic, nutraceutical and pharmaceutical fields [1]. Usually, peptides are obtained by means of solid phase synthesis which, unfortunately, doesn’t lead only to the main target molecule but to very complex mixtures [2]. The purification process is the bottleneck in the synthetic production of peptides, so it has to be optimized. Moreover, to purify industrial quantities of peptides, it is necessary to implement separation processes in a continuous manner, and this is feasible only knowing the optimal separation conditions in batch method. RP-LC is one of the most employed techniques for isolating the peptide of interest from the impurities and by-products deriving from synthesis [3].
In this work, a crude (=not purified) mixture of one octapeptide has been considered. Some preliminary gradient tests have been conducted to find out the best conditions for the separation of the peptide and the main impurity. Different mixtures of two mobile phases (0.02% TFA in MilliQ and 0.02% TFA in ACN) have been used during elution. Afterwards, the chromatographic profiles have been obtained also in isocratic conditions at different mobile phase compositions, chosen depending on gradient experiments. The shape of the profiles suggests that the adsorption isotherm is a convex upward type.
Using inverse method, an advanced numerical method which is particularly suitable when only small amounts of sample are available, experimental peaks have been fitted with different isotherm models; Langmuir isotherm gave the best results. The Langmuir isotherm parameters have been estimated for each fraction of organic modifier. It is known that the retention factor of a peptide, and therefore the isotherm parameters, are heavily affected by the organic solvent percentage [4]. This dependence has been evaluated; it has been found out that b (=adsorption equilibrium constant) follows an exponential trend and decreases if the organic modifier fraction increases.
[1] D. Agyei, C. M. Ongkudon, C. Y. Wei, A. S. Chan, M. K. Danquah, Bioprocess challenges to the isolation and purification of bioactive peptides. Food and Bioprod. Proc. 98 (2016) 244-256.
[2] S. Bernardi, D. Gétaz, N. Forrer, M. Morbidelli, Modeling of mixed-mode chromatography of peptides. Jour. Chrom. A 1283 (2013) 46-52.
[3] D. Gétaz, G. Stroelhein, A. Butté, M. Morbidelli, Model-based design of peptide chromatographic purification processes. Jour. Chrom. A 1284 (2013) 69-79.
[4] N. Marchetti, F. Dondi, A. Felinger, R. Guerrini, S. Salvadori, A. Cavazzini, Modeling of overloaded gradient elution of nociceptin/orphanin FQ in revesed-phase liquid chromatography. Jour. Chrom. A 1079 (2005) 162-172
Biomolecular NMR, a versatile tool for the understanding of protein science: retinoid-binding proteins as an example
No full textProtein science stands at the heart of modern life sciences because it unravels fundamental biological mechanisms and forms the basis for rapid advances in biomedicine and biotechnology. NMR spectroscopy is uniquely suited to study various aspects of protein structure, dynamics, molecular interactions and function, because information for individual residues can be obtained; moreover, kinetic data, low-populated states and the possible formation of intermediates on the reaction pathway can be determined.
The case of retinoid-binding proteins is discussed here, as an example. Vitamin A has diverse biological functions and is essential for human survival. It circulates in blood bound to serum retinol binding protein (RBP) and is transported into cells by a membrane receptor termed STRA6 (1). The cellular trafficking and metabolism of vitamin A are regulated primarily by specific high-affinity carriers called CRBP-I and CRBP-II. They represent an interesting case where structure determination as well as the study of fast dynamics (ps-ns time scale) (2) failed to elucidate the mode of retinol binding and thus to explain their diverse tissue distribution, functional roles and different ligand affinities.
The highly similar structure of the apo and holo forms (a beta-barrel with two short alpha-helices, see the cartoon) exhibits a closed conformation in both proteins, that seemingly offer no access for the ligand. Given the biological relevance of retinoids, the characterization of their protein interactions and targeted release is of special interest. To tackle this challenging subject we have employed a suite of NMR techniques: 15N relaxation dispersion experiments to investigate the proteins dynamics in the slower micros-ms timescale, line-shape analysis of 15N-HSQC spectra recorded during a retinol titration to get insights into the mechanism of ligand binding and H/D exchange experiments to investigate conformational stability. The results allowed to derive a model of retinol uptake, which is different for CRBP-I and CRBP-II (3, 4); moreover, a distinct local flexibility was found to modulate their binding properties (5).
The two proteins deliver retinol to microsomal membrane-bound enzymes, either for esterification with fatty acids (LRAT) (6, 7) or for oxidation to retinaldehyde (RDH) (8). Our current understanding of these processes remains incomplete, but there is evidence that the membrane microenvironment plays a role in the interactions of holo CRBPs with enzymes (8). To address this hypothesis, we have performed a series of NMR experiments with retinol-bound CRBP-I and CRBP-II in the presence of model membranes composed of either anionic or zwitterionic phospholipids, at varying protein:lipid molar ratios and ionic strength.
Both homologues interact with liposomes of anionic phospholipids, but in a significantly different way (9). A conformational rearrangement of the portal region, coupled to a change in protein dynamics, are required for retinol exchange; these processes seem to be triggered by a membrane-collision.
All the differences between CRBP-I and CRBP-II, when dissolved either in buffer or in the presence of biomembrane mimetic systems, may account for their distinct functional roles in the modulation of intracellular retinoid metabolism. Further experiments are in progress to better describe the ongoing processes in a biological context.
(1) Kawaguchi R., Yu J., Honda J., Hu J., Whitelegge J., Ping P., Wiita P., Bok D., Sun H. (2007) Science, 315, 820-825.
(2) Franzoni L., Lücke C., Pérez C., Cavazzini D., Rademacher M., Ludwig C., Spisni A., Rossi G.L., Rüterjans H. (2002) J. Biol. Chem., 277, 21983-21997.
(3) Mittag T., Franzoni L., Cavazzini D., Schaffhausen B., Rossi G.L., Günther U.L. (2006) J. Am. Chem. Soc., 128, 9844-9848.
(4) Franzoni L., Reed M., Cavazzini D., Rossi G.L., Günther U.L., in preparation.
(5) Franzoni L., Cavazzini D., Rossi G.L., Lücke C. (2010) J. Lipid Res., 51, 1332-1343.
(6) Amengual J., Golczak M., Palczewski K., von Lintig J. (2012) J. Biol. Chem., 287, 24216-24227.
(7) Jiang W., Napoli J.L. (2012) Biochim. Biophys. Acta, 1820, 859-869.
(8) Napoli J.L. (2012) Biochim. Biophys. Acta, 1821, 152-167.
(9) Franzoni L., Baroni F., Cavazzini D., Rossi G.L., Lücke C., in preparation
Post-processing methods of PIV instantaneous flow fields for unsteady flows in turbomachines
No full textThe Particle Image Velocimetry is undoubtedly one of the most important technique in Fluid-dynamics since it allows to obtain a direct and instantaneous visualization of the flow field in a non-intrusive way. This innovative technique spreads in a wide number of research fields, from aerodynamics to medicine, from biology to turbulence researches, from aerodynamics to combustion processes. The book is aimed at presenting the PIV technique and its wide range of possible applications so as to provide a reference for researchers who intended to exploit this innovative technique in their research fields. Several aspects and possible problems in the analysis of large- and micro-scale turbulent phenomena, two-phase flows and polymer melts, combustion processes and turbo-machinery flow fields, internal waves and river/ocean flows were considered
Teicoplanin Chiral Stationary Phases On 2.0 μm And 2.7 μm Superficially Porous Particles: Chromatographic Evaluation And Comparison With Teicoplanin On 1.9 μm Fully Porous Particles.
No full textFor the first time, a new generation of Chiral Stationary Phases (CSPs) for Ultra-High Performance Chromatography (UHPC) was prepared covalently bonding the teicoplanin (TE_A2) selector on Halo 2.0μm and 2.7μm (90Å, 125m2/g) Superficially Porous silica Particles (SPP) [1]. The new CSPs were compared with the already known UHPC-FPP-Titan-Tzwitt 1.9 CSP based on Fully Porous monodispersed silica Particles [2]. Columns with an internal diameter of 4.6 mm and different lengths (20, 50 and 100 mm) were packed with the three CSPs and characterized in terms of permeability, efficiency, retention and enantioselectivity under HILIC conditions. Van Deemter curves, generated using both achiral and chiral analytes, showed excellent results with more than 310000 plates/m at 1.6 mL/min (thiourea, k’= 0.6), and more than 290000 plates/m at 0.9 mL/min (2-(4-chloro-phenoxy)-propionic acid, 1st eluted enantiomer, k’= 0.8) on the SPP-Halo 2.0μm. The kinetic performance limits of the columns were investigated also through different kinetic plots, that proved the superior potential of the SPP-Halo 2.0μm and FPP-Titan 1.9μm compared to the SPP-Halo 2.7μm particles in the majority of the kinetic plots area, especially in the region of the ultra-fast/ultra-high efficiency separations. For several chiral analytes, the columns packed with SPP-Halo 2.0μm and FPP-Titan 1.9μm CSP were superior, in terms of efficiency, to the columns packed with SPP-Halo 2.7μm, proving the validity of the van Deemter analysis. Furthermore, from the thermodynamic point of view, the SPP-Halo 2.0μm have shown the best resolution power, with Rs values significantly higher than those of the SPP-Halo 2.7μm and FPP-Titan 1.9μm. Lastly Ultra-High speed – Ultra-High Performance applications performed on three 5-cm long columns, using chiral probes and different eluent flow-rate (1.0 – 6.0 mL/min) showed very high efficiency values and the FPP-Titan 1.9μm CSP, and again a lower performance for the columns packed with the SPP-Halo 2.7μm CSP. Considering the high resolution values observed, 2-cm long columns were prepared and used to explore Ultra-High speed – Ultra-High Performance separations in the seconds time regime: the enantiomers of Haloxyfop were resolved in only 3 seconds with a resolution Rs = 2 on the column packed with SPP-Halo 2.0μm CSP, using an eluent flow rate of 8.0 mL/min.
REFERENCES
1. O.H. Ismail, M. Antonelli, A. Ciogli, G. Sestito, D.S Bell, A. Cavazzini, C. Villani, F. Gasparrini, JCA, in preparation
2. O.H. Ismail, A. Ciogli, C. Villani, M. De Martino, M. Pierini, A. Cavazzini, D.S. Bell, F. Gasparrini, JCA, 1427 (2016), 55–6
Nouvelle édition de Charles Péguy, Note sur M. Bergson et Note conjointe sur M. Descartes
No full textCharles Péguy Note sur M. Bergson et Note conjointe sur M. Descartes par Andrea CAVAZZINI & Jonathan SOSKIN Liège, Presses Universitaires de Liège, 2016, 267 p. Avec les essais inédits de Andrea Cavazzini et Jonathan Soskin, Situations de Charles Péguy (pp. 5-19) et Le temps après l'éternité. Sur les Notes de Charles Péguy (pp. 199-258). Au-delà de l’apologie circonstancielle du bergsonisme contre ses adversaires rationalistes et thomistes, les deux textes testamentaires de Charles Péguy con..
Annonce de parution: Nouvelle édition de Charles Péguy, Note sur M. Bergson et Note conjointe sur M. Descartes
No full textCharles Péguy Note sur M. Bergson et Note conjointe sur M. Descartes par Andrea CAVAZZINI & Jonathan SOSKIN Liège, Presses Universitaires de Liège, 2016, 267 p. Avec les essais inédits de Andrea Cavazzini et Jonathan Soskin, Situations de Charles Péguy (pp. 5-19) et Le temps après l'éternité. Sur les Notes de Charles Péguy (pp. 199-258). Au-delà de l’apologie circonstancielle du bergsonisme contre ses adversaires rationalistes et thomistes, les deux textes testamentaires de Charles Péguy con..
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