Generic method for systematic phase selection and method development of biochromatographic processes. Part I. Selection of a suitable cation-exchanger for the purification of a pharmaceutical protein

Even if the first protein therapeutics are now for more than 20 years on the market the selection of suitable adsorbents for the preparative downstream processing (DSP) of these biomolecules as well as the method development towards process conditions are still based mainly on 'trial and error'. Therefore, theses processes are not perfectly efficient, but indeed very time consuming and laborious. In this study a novel systematic method is introduced to find a suitable adsorbent (not necessarily the best one) with appropriate separation parameters for a specific separation with reduced effort. Following this strategy, the adsorbents must first be packed into columns under preparative conditions and then characterized completely with regard to, e.g. pressure drop, k'-values, plate heights (HETP curves), selectivity and capacity by using test substances, which are similar in their characteristics (molecular mass, size, charge distribution, hydrophobicity) to the target proteins. With the database once determined, a preselection of most suitable adsorbents including separation parameters is made regarding chromatographic and also economical properties. After this, preparative experiments must be conducted with a reduced number of adsorbents to figure out the individual influence of side components. This approach is demonstrated for the separation of an exemplary industrial protein mixture using cation-exchange chromatography (CEX). Characterization of different weak CEX-adsorbents is illustrated. After comparing these phases with each other, a first preselection and a prediction of suitable adsorbents is made. In the following preparative separation conditions (load, velocity, gradient) are determined for the preparative separations using the database and results of some additional experiments. The final comparison of separation performance in preparative scale confirms this selection and so the applicability of the new method.     

Ionic Mobility, Phase Transitions, and Superionic Conduction in Solid Solutions (100 ? x)PbF2-xZrF4 and Crystals K2ZrF6, (NH4)2ZrF6, KSnZrF7, and M(NH4)6Zr4F23 (M = Li, Na)

Methods of (¹⁹F, ¹H) NMR and impedance spectroscopy are used to investigate the internal mobility and ionic conduction in solid solutions arising in the system PbF₂-ZrF₄ and polycrystals KSnZrF₇, Li(Na)(NH₄)₆Zr₄F₂₃, and M₂ZrF₆ (M = K, NH₄). Factors responsible for the form of ionic motions and their energetics at 170–550 K are considered. It is established that the phase transitions in these compounds are connected with the crystal transition to a superionic state and that the high ionic (superionic) conductivity of beta phases is due to the diffusion of fluoride ions, ammonium cations, and possibly alkali metal cations. The obtained data testify to a substantial role of chainlike aggregation of anionic groupings and a variableness of structural mechanisms of formation of such chains in fluorozirconates for the development of translational diffusion in these compounds.__________Translated from Elektrokhimiya, Vol. 41, No. 5, 2005, pp. 573–582.Original Russian Text Copyright © 2005 by Kavun, Uvarov, Slobodyuk, Goncharuk, Kotenkov, Tkachenko, Gerasimenko, Sergienko.     

From a point-like to an arbitrarily shaped sample: A survey of equations for electron paramagnetic resonance signal intensity calculations

A set of original, analytical equations useful for theoretical calculation of the electron paramagnetic resonance (EPR) signal intensity are presented for the multitude of sample shapes, which range from point-like, line-like, planar, rectangular, cubical, circular, cylindrical, spherical to an irregularly shaped sample. The samples can be situated at any available position within the prescribed part of the microwave cavity (a central cylinder of diameter 11 mm and length 23.5 mm, in either a Bruker single TE₁₀₂ or double TE₁₀₄ rectangular cavity, with the modulation coils situated in the left and right side cavity walls, which is connected to a X-band, field-modulated CW Bruker EPR spectrometer). The theoretical computations of EPR signal intensity can be used in the computer simulations in which: (i) the EPR signal intensity profiles are constructed; (ii) the optimal sample positions in the cavity to give a maximum value of signal intensity are found; (iii) the errors associated with sample positioning within the cavity when compared to a second sample of a different size, shape or position are studied.     

On the equation of state of classical one-component systems with long-range forces

Several definitions of the pressure are introduced for one-component systems and shown to be nonequivalent in the presence of a rigid neutralizing background. Relations between these pressures are derived for finite and infinite systems; these relations depend on the asymptotic behavior of the force at infinity, with the Coulomb force at the borderline between different properties. It is argued that only one of those definitions is physically acceptable and its properties are discussed in relation to the asymptotic behavior of the force. It is seen in particular that a knowledge of the state of the infinite system is not sufficient to determine its thermodynamic properties. The results are illustrated by some typical examples.For example, for two-dimensional systems with three-dimensional Coulomb interaction see refs. 2–4.     

Improving (Q)SAR predictions by examining bias in the selection of compounds for experimental testing

ABSTRACT

Existing data on structures and biological activities are limited and distributed unevenly across distinct molecular targets and chemical compounds. The question arises if these data represent an unbiased sample of the general population of chemical-biological interactions. To answer this question, we analyzed ChEMBL data for 87,583 molecules tested against 919 protein targets using supervised and unsupervised approaches. Hierarchical clustering of the Murcko frameworks generated using Chemistry Development Toolkit showed that the available data form a big diffuse cloud without apparent structure. In contrast hereto, PASS-based classifiers allowed prediction whether the compound had been tested against the particular molecular target, despite whether it was active or not. Thus, one may conclude that the selection of chemical compounds for testing against specific targets is biased, probably due to the influence of prior knowledge. We assessed the possibility to improve (Q)SAR predictions using this fact: PASS prediction of the interaction with the particular target for compounds predicted as tested against the target has significantly higher accuracy than for those predicted as untested (average ROC AUC are about 0.87 and 0.75, respectively). Thus, considering the existing bias in the data of the training set may increase the performance of virtual screening.     

Spectrophotometric resolution of ternary mixtures of pseudoephedrine hydrochloride, dextromethorphan hydrobromide, and sodium benzoate in syrups using wavelength selection by net analyte signals calculated with hybrid linear analysis

Hybrid linear analysis (HLA), as a recent factor-based multivariate calibration technique, was applied for the spectrophotometric determination of ternary mixtures of pseudoephedrine hydrochloride (PSU), dextromethorphan hydrobromide (DXT), and sodium benzoate (BNZ). The utilized HLA was assisted by a wavelength selection procedure which was based on the calculation of the net analyte signal (NAS) regression plot in any considered wavelengths window for each test sample, in addition to a moving window strategy for searching the region with maximum linearity of NAS regression plot (minimum error indicator (EI)). HLA was applied because it was simpler to adapt to the NAS regression plot methodology, and also used less factors than partial least squares (PLS). An orthogonal array design was applied for formation of calibration and prediction sets in the concentration ranges 0-7500 μmol L⁻¹ for PSU, 0-300 μmol L⁻¹ for DXT, and 0-1400 μmol L⁻¹ for BNZ. The method had the ability to select wavelength regions that minimize the effect of non-linearity of the spectral data, in addition to that of non-modeled interferences. The application of the selected wavelength regions improved the obtained relative standard error of predictions for PSU, DXT, and BNZ, respectively, from 5.24, 8.67, and 5.48% to 2.19, 5.21, and 3.62% (using lower number of factors). To check the ability of the proposed method in selection of linear regions of spectra, a test for detecting non-linear regions of spectral data in multivariate spectroscopic assays was also described. Additives in the commercial syrup samples did not interfere with their determinations. The method was successfully applied for the determination of pseudoephedrine HCl, dextromethorphan HBr, and sodium benzoate in cough suppressant syrup samples.     

Packings and stationary phases for biopolymer separations by HPLC

Summary Packings and stationary phases applied to high resolution separations of proteins, enzymes, and nucleic acids must satisfy a series of distinct criteria that are different from those usually required by HPLC of low molecular weight non-biologically active analytes. These requirements have been met through substantial improvements in classical gel media together with novel developments in silica supports, and have led to a family of products with tailor-made and reproducible properties. Supports consisting of cross-linked organic gels, and inorganic materials (mostly silicas) are now available with graduated particle sizes, pore sizes, porosities and surface areas as well as non-porous beads. A whole range of stationary phases, such as reversed phase, hydrophobic interaction, ion exchanger and affinity packings, were designed for application as chemical sensors for biopolymer recognition in adsorptive chromatography. The phase systems are operated in the gradient mode, giving high resolution and high peak capacities. In addition, aqueous liquid-liquid partitioning systems have been developed for the fractionation of proteins and nucleic acids. Size exclusion media complete the set of HPLC variants enabling a discrimination of proteins according to their size and shape in an isocratic elution mode. Basically, protein purification and isolation is a multistage process where-by the HPLC variants are combined in a logistic sequence, utilizing the different selectivities of the phase systems and thus maximising resolution, speed and throughput.     

Shape-controlled synthesis of metal nanostructures: the case of silver

The concept of shape-controlled synthesis is discussed by investigating the growth mechanisms for silver nanocubes, nanowires, and nanospheres produced through a polymer-mediated polyol process. Experimental parameters, such as the concentration of AgNO(3) (the precursor to silver), the molar ratio between poly(vinylpyrrolidone) (PVP, the capping agent) and AgNO(3), and the strength of chemical interaction between PVP and various crystallographic planes of silver, were found to determine the crystallinity of seeds (e.g., single crystal versus decahedral multiply twinned particles). In turn, the crystallinity of a seed and the extent of the PVP coverage on the seed were both instrumental in controlling the morphology of final product. The ability to generate silver nanostructures with well-defined morphologies provides a great opportunity to experimentally and systematically study the relationship between their properties and geometric shapes.