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Giorgio Carta

Publications and source records attributed to Giorgio Carta.

9 recordsLinked to original sources
PubMed2006

pH transitions in cation exchange chromatographic columns containing weak acid groups.

Complex pH transitions occur in cation exchange columns used for protein chromatography during equilibration and salt elution steps when the stationary phase contains weak acid groups even if the mobile phase is buffered and the buffering species do not interact with the stationary phase. In this work, we present a local equilibrium model to predict the magnitude and duration of these pH transients. The model equations are solved by the method of characteristics and by numerical simulations using an equilibrium-dispersive model. By incorporating an explicit description of the dissociation of the weakly ionogenic groups in the resin, we show that counterion binding in the column can be predicted for different buffer systems based on a single experimental resin titration curve without having to resort to empirically defined adsorption equilibrium constants. Model predictions based on these assumptions are found to be in excellent agreement with experimental results obtained for three different resins containing varying concentrations of weak acid groups. Four common buffer systems, acetate, citrate, MES, and phosphate are considered with both step and gradient changes in salt concentration at pH 5.5. Each buffer yields a different pH excursion behavior. We demonstrate that when the counterion concentration is kept constant in each of these buffers, which is needed to attain identical protein adsorption behavior, the magnitude of the pH transitions occurring during salt steps is nearly independent of the buffer system. On the other hand, the duration of the pH transitions is smallest for MES suggesting that this buffer system is preferable where pH variations are to be prevented.

Acetates↗
PubMed2006

Permuting input for more effective sampling of 3D conformer space.

SMILES strings and other classic 2D structural formats offer a convenient way to represent molecules as a simplistic connection table, with the inherent advantages of ease of handling and storage. In the context of virtual screening, chemical databases to be screened are often initially represented by canonicalised SMILES strings that can be filtered and pre-processed in a number of ways, resulting in molecules that occupy similar regions of chemical space to active compounds of a therapeutic target. A wide variety of software exists to convert molecules into SMILES format, namely, Mol2smi (Daylight Inc.), MOE (Chemical Computing Group) and Babel (Openeye Scientific Software). Depending on the algorithm employed, the atoms of a SMILES string defining a molecule can be ordered differently. Upon conversion to 3D coordinates they result in the production of ostensibly the same molecule. In this work we show how different permutations of a SMILES string can affect conformer generation, affecting reliability and repeatability of the results. Furthermore, we propose a novel procedure for the generation of conformers, taking advantage of the permutation of the input strings--both SMILES and other 2D formats, leading to more effective sampling of conformation space in output, and also implementing fingerprint and principal component analyses step to post process and visualise the results.

Algorithms↗
PubMed2006

Radiotracer measurements of protein mass transfer: kinetics in ion exchange media.

We describe a method to measure protein mass transfer kinetics in ion exchange adsorbents for preparative chromatography based on the use of radioactively labeled protein. The method was developed and evaluated using lysozyme as a test protein with the three commercial strong-acid cation exchangers SP-Sepharose-FF, SP-Sepharose-XL, and S-HyperD. Iodination with 125I was used to label the protein, which was added in trace amounts (approximately 0.1%) to an unlabeled protein solution. The solution was recirculated through a shallow bed of the adsorbent particles and the radioactivity accumulated in the bed measured with a gamma-counter as a function of time. Radiotracer-based kinetics measurements were found to be in good agreement with results obtained with a conventional shallow-bed technique, provided that freshly labeled protein solutions were used. The method has advantages in terms of simplicity, ability to deal with adsorption from complex mixtures, and the potential for measurements under tracer diffusion conditions. Kinetics results obtained for the three different stationary phases were generally consistent with previous studies. Protein mass transfer can be described by a pore diffusion model with a nearly salt-independent pore diffusivity for SP-Sepharose-FF and by a homogeneous diffusion model with a saltindependent adsorbed phase diffusivity for S-HyperD. However, it appears that a more complex model, accounting for parallel pore and surface diffusion, is needed to describe protein mass transfer in SP-Sepharose-XL. The modeling results were found to be correlated with the apparent pore sizes determined by inverse SEC.

Chromatography, Ion Exchange↗
PubMed2005

Two-component protein adsorption kinetics in porous ion exchange media.

This work provides a theoretical analysis of multicomponent adsorption kinetics for conditions typical of protein adsorption in porous ion exchangers as well as experimental results for the adsorption of lysozyme/cytochrome c mixtures in the cation exchanger SP-Sepharose-FF. The theory predicts the formation of overshoots in the intraparticle concentration profiles and in the total amount adsorbed for the more weakly adsorbed component. An analytical solution valid for the case where the isotherms are rectangular is developed and found to be in good agreement with the limiting behavior of the general numerical solution of the model equations. The experimental results show that the two proteins are competitively adsorbed and that an overshoot of adsorbed cytochrome c occurs during simultaneous adsorption. Model predictions based on the assumption that the adsorption isotherms are rectangular and that lysozyme completely displaces cytochrome c are in qualitative and quantitative agreement with the experimental kinetics suggesting that the overshoot phenomena observed with multicomponent systems in these resins can be explained with a diffusion model without the need to account for flux coupling or electrophoretic contributions to transport.

Adsorption↗
PubMed2005

Properties and performance of novel high-resolution/high-permeability ion-exchange media for protein chromatography.

There is continued interest in the development of stationary phases for protein chromatography that can provide high resolution at elevated flow rates of the mobile phase. When using porous particles, resolution and dynamic binding capacity decline rapidly as the flow rate is increased. Monolithic columns have been developed to overcome these limitations. However, there are difficulties in manufacturing homogeneous larger scale monoliths. In this paper we investigate the morphology and performance characteristics of columns based on new ion exchangers obtained by mechanically disrupting continuous beds of acrylamido-based polymeric media. Near colloidal suspensions of loose particles obtained with this procedure can be flow-packed in ordinary chromatography columns resulting in beds of unexpectedly high hydraulic permeability. Columns up to 2.2 cm in diameter were studied with both Q and S functionalized media. The hydraulic permeability and interparticle porosity of these columns were rather high. The permeabilities of the S and Q media were 1.5 x 10(-13) and 2.4 x 10(-13) m2, respectively, while the corresponding porosities were 60 and 70%. These porosity values are similar to those of monoliths, suggesting that these particles assemble under flow to give high-porosity bridged structures. The structure of these packed beds was further characterized by embedding small packed columns in resins and obtaining sections for microscopic observation. The sections reveal the presence of small aggregates of non-porous 1-3 microm particles, surrounded by flow channels several micrometers in size. The height equivalent to a theoretical plate under isocratic and gradient elution conditions and the dynamic binding capacity were determined for several proteins and were found to be virtually independent of flow.

Chromatography, Ion Exchange↗
PubMed2002

Protein adsorption on novel acrylamido-based polymeric ion-exchangers. IV. Effects of protein size on adsorption capacity and rate.

The effects of protein size on the adsorption capacity and rate is determined for an acrylamido-based polymeric anion-exchanger. The proteins lactalbumin, myoglobin, ovalbumin, BSA, conalbumin, IgG, and ferritin with molecular masses ranging from 15,000 to 450,000 were investigated. At high salt concentration (50 mM Tris-HCl containing 500 mM NaCl), only the smaller proteins lactalbumin and myoglobin gained access to a significant portion of the particle volume. The larger proteins were nearly completely excluded, in agreement with the results obtained for neutral macromolecules. By contrast, at low salt concentration (50 mM Tris-HCl), the adsorption capacity was very large (280-400 mg/ml of particle volume) for all the proteins studied except for ferritin, for which the capacity was much lower. This suggests that, provided the solute is not too large, the favorable electrostatic interaction overcomes the size exclusion effect. Adsorption rate measurements showed that mass transfer rates are also quite fast at low salt concentration. Effective diffusivities were determined by matching model and experimental results and were found to decrease substantially as the protein size increased. As previously observed, the homogeneous diffusion model was found to predict the experimentally observed trends with respect to protein concentration and boundary layer mass transfer effects.

Acrylamides↗
PubMedDate not supplied

Asymmetric synthesis with immobilized yeast in organic solvents: equilibrium conversion and effect of reactant partitioning on whole cell biocatalysis.

A newly isolated strain of the yeast Saccharomyces cerevisiae is investigated for the biocatalytic reduction of ketones and the oxidation of alcohols in organic solvents. The yeast cells are immobilized by entrapment within calcium alginate beads and are found to possess the ability to stereoselectively reduce prochiral ketones and oxidize chiral alcohols to equilibrium conversions. The effect of reactant partitioning on the initial rate of the reactions is also investigated. The observed initial rates are found to vary inversely with reactant partitioning between the organic solvent and the biocatalyst beads. A kinetic model is developed to describe the initial reaction rate of hexanone reduction as a function of substrate concentration within the alginate beads.

Alcohols↗
PubMedDate not supplied

Considerations in compound database preparation--"hidden" impact on virtual screening results.

Structure-based virtual screening (SBVS) utilizing docking algorithms has become an essential tool in the drug discovery process, and significant progress has been made in successfully applying the technique to a wide range of receptor targets. In silico validation of virtual screening protocols before application to a receptor target using a corporate or commercially available compound collection is key to establishing a successful process. Ultimately, retrieval of a set of active compounds from a database of inactives is required, and the metric of enrichment (E) is habitually used to discern the quality of separation of the two. Numerous reports have addressed the performance of docking algorithms with regard to the quality of binding mode prediction and the issue of postprocessing "hit lists" of docked ligands. However, the impact of ligand database preprocessing has yet to be examined in the context of virtual screening and prioritization of compounds for biological evaluation. We provide an insight into the implications of cheminformatic preprocessing of a validation database of compounds where multiple protonated, tautomeric, stereochemical, and conformational states have been enumerated. Several commonly used methods for the generation of ligand conformations and conformational ensembles are examined, paired with an exhaustive rigid-body algorithm for the docking of different "multimeric" compound representations to the ligand binding site of the human estrogen receptor alpha. Chemgauss, a shapegaussian scoring function with intrinsic chemical knowledge, was combined with PLP as a consensus-scoring scheme to rank output from the docking protocol and enrichment rates calculated for each screen. The overheads of CPU consumption and the effect on relative database size (disk requirement) for each of the protocols employed are considered. Assessment of these parameters indicates that SBVS enrichments are highly dependent on the initial cheminformatic treatment(s) used in database construction. The interplay of SMILES representations, stereochemical information, protonation state enumeration, and ligand conformation ensembles are critical in achieving optimum enrichment rates in such screening.

Computer Simulation↗