Ruth Freitag

Comparison of polymer coatings of capillaries for capillary electrophoresis with respect to their applicability to molecular imprinting and electrochromatography

Abstract In molecular imprinting (MI), interactive monomers and suitable cross-linking agents are polymerized in the presence of a template. Once the template has been removed, the remaining space acts as a highly specific binding site for the template or analogs thereof, due to the unique three-dimensional arrangement of interaction points. Several steps are involved in producing such a polymer coat inside a capillary electrophoresis capillary. First, the silanization of the inner surface of the capillary with a suitable silane is necessary, to link a monolayer of unsaturated groups suitable for polymerization to the capillary surface. These monomeric groups are then integrated into the three-dimensional polymer coat produced in the next step. MI-capillary coatings ideally are highly porous and of a thickness, δ, which is smaller than the inner radius, r, of the capillary in question. Porous polymer networks can be produced by dispersive polymerization using a suitable solvent (porogen). However, the exact conditions for producing a coating suitable for capillary zone electrophoresis had to be determined experimentally. Seven porogens, namely hexane, toluene, tetrahydrofuran, acetonitrile, CHCl3, dimethyl sulfoxide and dimethylformamide, and two cross-linkers, namely ethyleneglycoldimethacrylate and divinylbenzene, at concentrations of between 5 and 20% (v/v) were investigated. In about 20% of the combinations, a polymer coat of the desired qualities was obtained. The applicability of the MI capillaries to specific separations was demonstrated for the separation of a racemic mixture of S(+)- and R(−)-2-phenylpropionic acid. trans-3(3-Pyridyl)-acrylic acid was used as the interactive monomer in this case.

Acrylamide-based monoliths as robust stationary phases for capillary electrochromatography.

A method is described for the synthesis of rigid, macroporous polymers (monoliths) to be used as stationary phases in capillary electrochromatography (CEC). The procedure reproducibly results in columns with good mechanical and chemical stability. Once the procedure was optimized, it yielded the desired CEC columns in nearly 100% of the cases. The batch-to-batch standard deviation of the migration of the electroosmotic flow (EOF) marker for nine randomly chosen columns was 5%. The polymerization is carried out inside the capillary, an aqueous phase is used as solvent. Monomers based on acrylamides with varying hydrophilicity were used to introduce the interactive moieties together with piperazine diacrylamide as cross-linker and vinylsulfonic acid as provider of the charged, EOF-producing moieties. The pore size of the monoliths was adjusted by adding varying amounts of ammonium sulfate to the reaction mixture. In this manner, the average pore size of a given monolith could be reproducibly adjusted to values ranging from 50 nm to 1.3 microm. The procedure was optimized for four particular types of monoliths, which differed in hydrophobicity. The latter was adjusted by introducing suitable co-monomers, such as alkyl chain-bearing molecules, into the monolithic structure. Attempts to systematically investigate the chromatographic behavior of the monolithic stationary phases were made, using a model mixture of aromatic compounds as sample. The standard deviations for the run-to-run reproducibility of the retention times for unretained and retained analytes were <1.5%. Flat Van Deemter curves were measured even at elevated flow-rates (2 mm/s). Plate heights between 10 and 15 microm were measured in this range. The retention order was taken as the principal indication for the chromatographic mode. The separation was found to be governed neither by pure reversed-phase nor by pure normal-phase chromatography, even on monoliths, where large amounts of C6 ligands had been introduced.

Influence of Polymer Architecture and Molecular Weight of Poly(2-(dimethylamino)ethyl methacrylate) Polycations on Transfection Efficiency and Cell Viability in Gene Delivery

Nonviral gene delivery with the help of polycations has raised considerable interest in the scientific community over the past decades. Herein, we present a systematic study on the influence of the molecular weight and architecture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) on the transfection efficiency and the cytotoxicity in CHO-K1 cells. A library of well-defined homopolymers with a linear and star-shaped topology (3- and 5-arm stars) was synthesized via atom transfer radical polymerization (ATRP). The molecular weights of the polycations ranged from 16 to 158 kDa. We found that the cytotoxicity at a given molecular weight decreased with increasing number of arms. For a successful transfection a minimum molecular weight was necessary, since the polymers with a number-average molecular weight, M(n), below 20 kDa showed negligible transfection efficiency at any of the tested polyelectrolyte complex compositions. From the combined analysis of cytotoxicity and transfection data, we propose that polymers with a branched architecture and an intermediate molecular weight are the most promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable transfection results.

Investigation of the LCST of polyacrylamides as a function of molecular parameters and the solvent composition

The paper investigates the thermoprecipitation of two macromolecule structures, poly(N-isopropylacrylamide) (poly-NIPA) and poly(N,N-diethylacrylamide) (poly-DEA) from aqueous solution. The majority of the data are collected for small (M w < 5000 g/mol) homogeneous (D < 1.3) molecules of the indicated type synthesized by anionic, group transfer, and radical polymerization in the presence of a chain transfer agent. Conventional radical polymers (M w < 200,000 g/mol) are also synthesized and used for comparison. Turbidity curves (photometry) and transition enthalpies (high sensitivity differential scanning calorimetry) are measured to investigate the phase transition as a function of the molecular size and the tacticity as well as the concentration of certain solution additives (simple salts, glucose, and the surfactant tetrabutylammonium acetate) and mixtures thereof. Where applicable, the results are interpreted on the basis of a two-state model to gain insight in the cooperativity of the transition.

An introduction to monolithic disks as stationary phases for high performance biochromatography

Monolithic stationary phases have revolutionized protein chromatography because they combine speed, capacity, and resolution in a unique manner. Since such stationary phases contain no particles but only flow-through pores, the usual mass transfer restrictions to the chromatography of large molecules are not observed and extremely fast separations become possible. Recently the area of application of monolith chromatography has been extended to the separation and analysis of small molecules and plasmid DNA. This review summarizes the state of art in high performance monolith and especially high performance monolithic disk chromatography (HPMDC). The current understanding of the theory of protein HPMDC is summarized, while an introduction to the evolving field of small molecule HPMDC is attempted. The basic differences between the monolithic disks and columns packed with conventional stationary phases (including perfusion and micropellicular particles) but also monolithic columns (porous rods) are outlined. Finally, the potential of HPMDC to analytical and preparative biochromatography is demonstrated by a discussion of recent applications of chromatographic disks for protein isolation and bioprocess analysis.

Protein purification by affinity precipitation

Developing the most efficient strategy for the purification of a (recombinant) protein especially at large scale remains a challenge. A typical problem of the downstream process of mammalian cell products is, for instance, the early capture of the highly diluted product from the complex process stream. Affinity precipitation has been suggested in this context. The technique is known for over 20 years, but has recently received more attention due to the development of new materials for its implementation, but also because it seems ideally suited to specific product capture at large scale. The present review gives a comprehensive overview over this technique. Besides an introduction to the basic principle and a brief summary of the historical development, the main focus is on the current state-of-art of the technique, the available materials, important recent applications, as well as process design strategies and operating procedures. Special consideration is given to affinity precipitation for product recovery at large scale.

Dual-responsive magnetic core-shell nanoparticles for nonviral gene delivery and cell separation.

We present the synthesis of dual-responsive (pH and temperature) magnetic core-shell nanoparticles utilizing the grafting-from approach. First, oleic acid stabilized superparamagnetic maghemite (γ-Fe(2)O(3)) nanoparticles (NPs), prepared by thermal decomposition of iron pentacarbonyl, were surface-functionalized with ATRP initiating sites bearing a dopamine anchor group via ligand exchange. Subsequently, 2-(dimethylamino)ethyl methacrylate (DMAEMA) was polymerized from the surface by ATRP, yielding dual-responsive magnetic core-shell NPs (γ-Fe(2)O(3)@PDMAEMA). The attachment of the dopamine anchor group on the nanoparticles surface is shown to be reversible to a certain extent, resulting in a grafting density of 0.15 chains per nm(2) after purification. Nevertheless, the grafted NPs show excellent long-term stability in water over a wide pH range and exhibit a pH- and temperature-dependent reversible agglomeration, as revealed by turbidimetry. The efficiency of γ-Fe(2)O(3)@PDMAEMA hybrid nanoparticles as a potential transfection agent was explored under standard conditions in CHO-K1 cells. Remarkably, γ-Fe(2)O(3)@PDMAEMA led to a 2-fold increase in the transfection efficiency without increasing the cytotoxicity, as compared to polyethyleneimine (PEI), and yielded on average more than 50% transfected cells. Moreover, after transfection with the hybrid nanoparticles, the cells acquired magnetic properties that could be used for selective isolation of transfected cells.

Comparison of antibody binding to immobilized group specific affinity ligands in high performance monolith affinity chromatography

A novel biochromatographic principle is introduced taking the quantitative analysis of affinity interactions between antibodies and immobilized group specific ligands (protein A, G, and L) as example. The name high performance monolith affinity chromatography (HPMAC) is proposed for this technique. HPMAC uses rigid, macroporous monoliths, so-called convective interaction media (CIM)-disks, as stationary phase. An optimized procedure is described for the covalent immobilization of the group specific affinity ligands to such disks. The binding of polyclonal bovine IgG and a recombinant human antibody (type IgGl-kappa) to all affinity disks is discussed. An essential feature of HPMAC is its compatibility to unusually high mobile phase flow rates ( > 4 ml/min). Chromatographic experiments are thus completed within seconds without significant loss in binding capacity and retentive power. This makes HPMAC a promising tool for applications in fast process monitoring or screening. As an example for the former, the direct quantitative isolation of recombinant antibodies from serum-free culture supernatant is demonstrated.

Fast isolation of protein receptors from streptococci G by means of macroporous affinity discs

A fast affinity method for the semi-preparative isolation of recombinant Protein G from E. coli cell lysate is proposed. Rigid, macroporous affinity discs based on a glycidyl methacrylate-co-ethylene dimethacrylate polymer were used as chromatographic supports. The specific ligands (here human immunoglobulin G, hIgG) were immobilized by the one-step reaction between native epoxy groups of the polymer surface and epsilon-amino groups of the IgG molecules. No intermediate spacer was necessary to reach full biological activity of the ligand. The globular affinity ligands are located directly on the pore wall surface and are thereby freely accessible to target molecules (here Protein G) migrating with the mobile phase through the pores. It is shown that the conditions chosen for the hIgG immobilization do not involve an active site of the protein and thus do not bias the formation of the affinity complex. Chromatographically determined constants of dissociation of hIgG-Protein G affinity complexes confirm the high selectivity of this separation method. Two different aspects of the affinity separation are discussed, which differ mostly in terms of scale. In disc chromatography, high volumetric flow velocities are possible because of the small backpressure. Since in addition the mass transfer is more efficient, it becomes possible to achieve very short analysis times. The discs proposed can be used in a single-step enrichment of Protein G from lysates of non-pathogenic E. coli. Gel electrophoresis data are used to demonstrate the high degree of purity achieved for the final product.

Immobilized metal affinity membrane adsorbers as stationary phases for metal interaction protein separation

Abstract Novel immobilized metal affinity membrane adsorbers (IMA-MA) were studied for potential use as stationary phases for protein separation. Protein adsorption on IMA-MA loaded with Cu(II), Ni(II), ZN(II) and Co(II) ions was compared as a function of the flow-rate and the ionic strength of the elution buffer. To exclude the possibility of mixed-mode interaction in the experiments, the binding of proteins similar in terms of hydrophobicity, isoelectric point, size and mass-to-charge ratio but differing in their number of surface histidine residues was investigated. Matrix-assisted laser desorption/ionization mass spectrometry was used to distinguish between these proteins in the eluted fractions. Salt concentration of at least 0.5 M NACl and flow-rates below 2 ml min−1 were found suitable to ensure an adsorption mechanism based on affinity interaction between the proteins and the chelated metal ions. In an application study, the IMA-MA and conventional chelating Sepharose fast flow columns were compared for the isolation of a recombinant fusion protein (EcoR V), which carried a polyhistidine sequence (HIS6-tag) at the N-terminus.