Promiscuity of host cell proteins in the purification of histidine tagged recombinant xylanase A by IMAC procedures: A case study with a Ni2+-tacn-based IMAC system.

Determination of the extent of host cell protein (HCP) contamination is an essential pre-requisite to validate the chromatographic purification of recombinant proteins. This study explores how different experimental conditions affect the HCP profiles generated during the immobilised metal ion affinity chromatographic (IMAC) purification with a Ni2+-1,4,7-triaza-cyclononane (tacn) Sepharose FF™ sorbent of the Bacillus halodurans N- and C-terminal His6-tagged xylanase A, expressed by Escherichia coli BL21(DE3) cells, and captured directly from cell lysates. Comparative studies were also carried out under identical loading, wash and elution conditions using nitrilotriacetic acid (NTA), also immobilised onto an agarose support and complexed with Ni2+ ions. High-resolution tandem mass spectrometry of the tryptic peptides derived from the proteins present in the IMAC flow-through, wash and elution fractions confirmed that the E. coli BL21(DE3) HCP profiles were dependent on the choice of adsorbent. With feedstocks containing the N- or C-terminal His6-tagged xylanase A, in several instances the same E. coli BL21(DE3) HCPs were found to co-elute with the tagged protein from either adsorbent, indicating a preferential ability of some HCPs to bind to both the IMAC resin and to the recombinant protein. This promiscuous behaviour has been found to be due to factors other than just the presence of histidine-rich motifs within the amino acid sequences of these HCPs. This case study demonstrates that the choice of protein expression and separation conditions impact on the levels of HCP contamination when different IMAC systems are employed.

Advances in the development of molecularly imprinted polymers for the separation and analysis of proteins with liquid chromatography.

This review documents recent advances in the design, synthesis, characterization, and application of molecularly imprinted polymers in the form of monoliths and particles/beads for the use in the separation and analysis of proteins with solid-phase extraction or liquid chromatography. The merits of three-dimensional molecular imprinting, whereby the molecular template is randomly embedded in the polymer, and two-dimensional imprinting, in which the template is confined to the surface, are described. Target protein binding can be achieved by either using the entire protein as a template or by using a protein substructure as template, that is, a peptide, as in the "epitope" approach. The intended approach and strategy then determine the choice of polymerization method. A synopsis has been provided on methods used for the physical, chemical, and functional characterizations and associated performance evaluations of molecularly imprinted and nonimprinted control polymers, involving a diverse range of analytical techniques commonly used for low and high molecular mass analytes. Examples of recent applications demonstrate that, due to the versatility of imprinting methods, molecularly imprinted monoliths or particles/beads can be adapted to protein extraction/depletion and separation procedures relevant to, for example, protein biomarker detection and quantification in biomedical diagnostics and targeted proteomics.

Use of peak sharpening effects to improve the separation of chiral compounds with molecularly imprinted porous polymer layer open-tubular capillaries.

This investigation demonstrates the application of a new peak sharpening technique to improve the separation of difficult-to-resolve racemic mixtures in capillary electro-chromatography. Molecularly imprinted porous layer open tubular (MIP-PLOT) capillaries, prepared by a layer-on-layer polymerization approach with Z-l-Asp-OH as the template, were selected to validate the approach. SEM revealed that the polymer film thickness can be varied by changes in both the polymer composition and the layer-on-layer regime. Capillaries made with methacrylic acid as the functional monomer could not separate the Z-Asp-OH racemate, due to weak interactions between the MIP-PLOT material and the target analytes. In contrast, MIP-PLOT capillaries prepared with 4-vinylpyridine as the functional monomer resulted in increased ionic interactions with the target analytes. Separation of the enantiomers could be enhanced when a peak zone sharpening effect was exploited through the use of specific BGE compositions and by taking advantage of eigenpeak phenomena. In this manner, the position of a sharpening zone and the peak shape of the sample analytes could be fine-tuned, so that when the sharpening zone and the target analyte co-migrated the separation of the Z-l-Asp-OH enantiomer from its d-enantiomer in a racemic mixture could be achieved under overloading conditions.

Molecularly imprinted polymer membranes and thin films for the separation and sensing of biomacromolecules.

This review describes recent advances associated with the development of surface imprinting methods for the synthesis of polymeric membranes and thin films, which possess the capability to selectively and specifically recognize biomacromolecules, such as proteins and single- and double-stranded DNA, employing "epitope" or "whole molecule" approaches. Synthetic procedures to create different molecularly imprinted polymer membranes or thin films are discussed, including grafting/in situ polymerization, drop-, dip-, or spin-coating procedures, electropolymerization as well as micro-contact or stamp lithography imprinting methods. Highly sensitive techniques for surface characterization and analyte detection are described, encompassing luminescence and fluorescence spectroscopy, X-ray photoelectron spectroscopy, FTIR spectroscopy, surface-enhanced Raman spectroscopy, atomic force microscopy, quartz crystal microbalance analysis, cyclic voltammetry, and surface plasmon resonance. These developments are providing new avenues to produce bioelectronic sensors and new ways to explore through advanced separation science procedures complex phenomena associated with the origins of biorecognition in nature.

Contribution of Eigenmobility Shifts to the Separation of Peptides in Capillary Electrophoresis with Aqueous-Acetonitrile Background Electrolytes.

In this investigation, the mobility of system eigenpeaks in capillary electrophoresis (CE) was experimentally found to decrease when the background electrolyte (BGE) contained higher percentages of acetonitrile. In order to explain this observation, the effects of changes in the pH and ionic strength of the BGE on the pKa and actual mobility of each constituent in the system were determined, and the results evaluated in terms of their theoretical basis. Utilizing the derived values of each of these parameters, the software Peakmaster was then applied to simulate the eigenpeak mobility. Although general trends for BGEs with different acetonitrile contents could be simulated, these simulations did not exactly match the experimental results. To account for this divergence between theory and experimental practice, the consequences of tube radial distribution of the organic solvent in an aqueous-organic system within the capillary and the effects of radial ion distribution leading to the electro-osmotic flow mobility (EOF) are proposed to be the cause of this deviation. Consequently, the Debye-Hückel approximation and Boltzmann distribution function were employed to calculate the amount of each constituent across the radius of the capillary. The inhomogeneous radial distributions of the constituents in the BGE and the organic solvent were simplified to a 1-dimensional problem based on a 4-constituent BGE approximation. A high level of correlation was then achieved between the experimental results and the corresponding CE separations simulated using Peakmaster. In addition, cancellation or suppression of the peak broadening was experimentally and theoretically demonstrated by taking advantage of the influence of a second independent system eigenpeak. The outcome from these studies was a new way to achieve sharpening of specific peaks in the CE separations of peptides.

Studies on the binding sites of IgG2 monoclonal antibodies recognized by terpyridine-based affinity ligands.

This investigation has examined the origin of the molecular recognition associated with the interaction of monoclonal IgG2's with terpyridine-based ligands immobilized onto agarose-derived chromatographic adsorbents. Isothermal titration calorimetric (ITC) methods have been employed to acquire thermodynamic data associated with the IgG2-ligand binding. These ITC investigations have documented that different enthalpic and entropic processes are involved depending on the nature of the chemical substituents in the core structure of the terpyridinyl moiety. In addition, molecular docking studies have been carried out with IgG2 structures with the objective to identify possible ligand binding sites and key interacting amino acid residues. These molecular docking experiments with the different terpyridine-based ligands have shown that all of the examined ligands can potentially undergo favorable interactions with a site located within the Fab region of the IgG2. However, another favorable binding site was also identified from the docking poses to exist within the Fc region of the IgG2 for some, but not all, of the ligands studied. These investigations have provided a basis to elucidate the unique binding properties and chromatographic behaviors shown by several substituted terpyridine ligands in their interaction with IgGs of different isotype. Copyright © 2016 John Wiley & Sons, Ltd.

Open tubular-capillary electrochromatography: Developments and applications from 2013 to 2015.

Open tubular CEC (OT-CEC) separates analyte mixtures by a combination of electrophoretic, electro-osmotic, and/or chromatographic effects. OT-CEC research is an active and growing field, with studies encompassing a wide range of investigations related to new strategies for chemical modification of the inner surface of the capillary, leading to the introduction of novel stationary phase coatings. This review has examined the literature on OT-CEC from 2013 to August 2015 and highlights the developments in the fabrication of highly selective stationary phases, based on materials that include cyclodextrin chiral selectors, graphene and graphene oxide, metal-organic frameworks, molecularly imprinted polymers, nanoparticles, nanolatex particles, nanocomposites, in situ generated polymers, block polymers, tentacle-type polymers, polyelectrolyte multilayers, polysaccharides, phospholipids, and proteins. This review, while considering the development of novel OT-CEC coating materials, specifically examines different immobilization or coating methodologies and approaches and also discusses the separation mechanisms that occur with these new materials. These OT-CEC coatings are intended mainly to separate low molecular weight molecules relevant to the pharmaceutical, agricultural, and food industries as well as for use in environmental monitoring.

On-line determination by small angle X-ray scattering of the shape of hen egg white lysozyme immediately following elution from a hydrophobic interaction chromatography column.

This study documents the use of an integrated approach, involving on-line hydrophobic interaction chromatography interfaced with Small Angle X-ray Scattering (HIC-SAXS) measurements, to monitor the conformational status of proteins immediately upon elution from a chromatographic column operated at different temperatures. Moreover, this approach provides an additional avenue to interrogate the changes in protein shape that may occur across the eluted chromatographic peak. To this end, radii of gyration were extrapolated from the Guinier approximation with the HIC-SAXS data, whilst pair distribution functions and bead model simulations were generated by using the indirect transform program GNOM and ab initio reconstruction with GASBOR to provide further insight into protein conformational changes that occur during hydrophobic interaction chromatography.

Impact on monoclonal antibody production in murine hybridoma cell cultures of adenosine receptor antagonists and phosphodiesterase inhibitors.

The effects of different adenosine receptor antagonists and cyclic nucleotide phosphodiesterase (PDE) inhibitors on monoclonal antibody (mAb) titer and cell viability of murine hybridoma cells in culture were measured as part of our investigations to discover additives that enhance mAb production. Specific adenosine receptor antagonists and PDE inhibitors were found to enhance or decrease the titer of immunoglobulin G1 (IgG1) mAbs relative to negative controls, depending on the specific compound and cell line employed. The observed enhancements or decreases in IgG1 mAb titer appeared to be mainly due to an increase or decrease in specific productivity rates (ngmAb/cell), respectively. The different effects of the selective adenosine antagonists suggest that antagonism at the level of the adenosine A2A and A1 or the adenosine A3 receptors result in either enhancement or suppression of IgG1 mAb production by hybridoma cells. Overall, these studies have identified hitherto unknown activities of specific adenosine antagonists and PDE inhibitors which indicate they may have valuable roles as cell culture additives in industrial biomanufacturing processes designed to enhance the yields of mAbs or other recombinant proteins produced by mammalian cell culture procedures.

The Application of Template Selectophores for the Preparation of Molecularly Imprinted Polymers.

Molecularly imprinted polymers are versatile materials with wide application scope for the detection, capture and separation of specific compounds present in complex feed stocks. A major challenge associated with their preparation has been the need to sacrifice one mole equivalent of the template molecule to generate the complementary polymer cavities that selectively bind the target molecule. Moreover, template molecules can often be difficult to synthesise, expensive or lack stability. In this study, we describe a new approach, directed at the use of synthetic selectophores, chosen as readily prepared and low cost structural analogues with recognition groups in similar three-dimensional arrangements as found in the target molecule. To validate the approach, a comparative study of selectophores related to the polyphenolic compound (E)-resveratrol has been undertaken using traditional and green chemical synthetic approaches. These molecular mimic compounds were employed as polymer templates and also as binding analytes to interrogate the recognition sites associated with the molecularly imprinted polymers. Importantly, the study confirms that the use of selectophores has the potential to confer practical advantages, including access to more efficient methods for selection and preparation of suitable template molecules with a broader range of molecular diversity, as well as delivering imprinted polymers capable of recognizing the target compound and structurally related products.

Miniaturized molecularly imprinted polymer extraction method for the gas chromatographic analysis of flavonoids.

In this study, the use of monolithic molecularly imprinted polymers in a micropipette tip format allowing the simple and fast extraction of flavonoids from standard solutions and a black tea sample is demonstrated. The imprinted polymer employed quercetin, methacrylic acid or 4-vinylpyridine, and ethylene glycol dimethacrylate as template, functional monomer, and cross-linker, respectively. Surface morphologies of the quercetin-imprinted polymers and the corresponding nonimprinted polymers were characterized by SEM. Extraction of flavonoid standards was performed to evaluate the selectivity and recovery with these imprinted and nonimprinted polymers. Flavonoid compositions in aliquots eluted from the tips were identified using fast GC with flame ionization detection. Maximum specific capacities of 0.2, 5.7, and 16.0 mg/g for catechin, morin, and quercetin, respectively, were obtained with the imprinted polymer prepared with methacrylic acid, with the corresponding recoveries of 99.8, 98.8, and 95.4%, respectively. Efficient extraction by the quercetin-imprinted polymer of epicatechin, catechin, and quercetin from an apple-flavored black tea sample was achieved, with GC-MS employed for compound identification for both the tea and extracted samples.

Precipitation of iron on the surface of Leptospira interrogans is associated with mutation of the stress response metalloprotease HtpX.

High concentrations of free metal ions in the environment can be detrimental to bacterial survival. However, bacteria utilize strategies, including the activation of stress response pathways and immobilizing chemical elements on their surface, to limit this toxicity. In this study, we characterized LA4131, the HtpX-like M48 metalloprotease from Leptospira interrogans, with a putative role in bacterial stress response and membrane homeostasis. Growth of the la4131 transposon mutant strain (L522) in 360 µM FeSO4 (10-fold the normal in vitro concentration) resulted in the production of an amorphous iron precipitate. Atomic force microscopy and transmission electron microscopy analysis of the strain demonstrated that precipitate production was associated with the generation and release of outer membrane vesicles (OMVs) from the leptospiral surface. Transcriptional studies indicated that inactivation of la4131 resulted in altered expression of a subset of metal toxicity and stress response genes. Combining these findings, this report describes OMV production in response to environmental stressors and associates OMV production with the in vitro activity of an HtpX-like metalloprotease.

Selectivity differences of water-soluble vitamins separated on hydrophilic interaction stationary phases.

In this study, the retention behavior and selectivity differences of water-soluble vitamins were evaluated with three types of polar stationary phases (i.e. an underivatized silica phase, an amide phase, and an amino phase) operated in the hydrophilic interaction chromatographic mode with ESI mass spectrometric detection. The effects of mobile phase composition, including buffer pH and concentration, on the retention and selectivity of the vitamins were investigated. In all stationary phases, the neutral or weakly charged vitamins exhibited very weak retention under each of the pH conditions, while the acidic and more basic vitamins showed diverse retention behaviors. With the underivatized silica phase, increasing the salt concentration of the mobile phase resulted in enhanced retention of the acidic vitamins, but decreased retention of the basic vitamins. These observations thus signify the involvement of secondary mechanisms, such as electrostatic interaction in the retention of these analytes. Under optimized conditions, a baseline separation of all vitamins was achieved with excellent peak efficiency. In addition, the effects of water content in the sample on retention and peak efficiency were examined, with sample stacking effects observed when the injected sample contained a high amount of water.

Simultaneous separation of hydrophobic and polar bases using a silica hydride stationary phase.

In this study, the retention behavior of selected hydrophobic and polar bases on a minimally modified silica hydride phase was investigated. From these results and the associated retention plots, significant differences in the chromatographic dependencies of these two classes of basic compounds were evident. The polar bases exhibited strong retention with mobile phases of high organic solvent content, but displayed weak retention with mobile phases of high water content. In contrast, the hydrophobic bases showed "U-shape" retention dependencies, indicative of the interplay of both RP and normal-phase retention characteristics. These studies have demonstrated that hydrophobic and polar bases can be simultaneously separated on the same column either under typical RP-like or aqueous normal-phase-like conditions, respectively, with distinctive selectivity. Finally, the effects of temperature on the RP and aqueous normal phase modality of separations with these analytes were investigated, where discrete changes in retention behavior were also observed.

Analysis of polar peptides using a silica hydride column and high aqueous content mobile phases.

The retention behavior of a set of polar peptides separated on a silica hydride stationary phase was examined with a capillary HPLC system coupled to ESI-MS detection. The mobile phases consisted of formic acid or acetic acid/acetonitrile/water mixtures with the acetonitrile content ranging from 5 to 80% v/v. The effects on peptide retention of these two acidic buffer additives and their concentrations in the mobile phase were systematically investigated. Strong retention of the peptides on the silica hydride phase was observed with relatively high-organic low-aqueous mobile phases (i.e. under aqueous normal-phase conditions). However, when low concentrations of acetic acid were employed as the buffer additive, strong retention of the peptides was also observed even when high aqueous content mobile phases were employed. This unique feature of the stationary phase therefore provides an opportunity for chromatographic analysis of polar peptides with water-rich eluents, a feature usually not feasible with traditional RP sorbents, and thus under conditions more compatible with analytical green chemistry criteria. In addition, both isocratic and gradient elution procedures can be employed to optimize peptide separations with excellent reproducibility and resolution under these high aqueous mobile phase conditions with this silica hydride stationary phase.

Rapid microwave-assisted chemical cleavage - mass spectrometric method for the identification of hemoglobin variants in blood.

A sensitive, rapid analytical method has been developed for the characterization of human hemoglobin disorders with very small volumes (<1 µL) of blood. As an alternative to conventional enzymatic digestion, a site-specific chemical cleavage method has been established using 0.05% formic acid under microwave-irradiation conditions for short time intervals, for example, less than 10 min. Peptide analysis was performed by MALDI TOF MS and capillary liquid chromatographic ESI MS/MS. The cleavage of the hemoglobin chains with formic acid occurred at either side or at both sides (C- and N-terminal) of aspartic acid residues, but preferentially N-terminally. The method has been applied to blood samples from hemoglobin S carrier heterozygotes and hemoglobin S thalassaemia compound heterozygotes with a reduced expression level of hemoglobin S. Both MALDI TOF MS and ESI MS/MS analysis allowed the identification of the hemoglobin S "signature" peptide. This alternative method of sample preparation is compatible with MS techniques and is expected to significantly contribute to the further development of rapid, robust, reproducible and sensitive analytical methods in proteomics and biomedical diagnostics where protein variant characterization is a crucial factor for biomarker discovery and disease identification.

Batch binding studies with thermo-responsive polymer grafted sepharose 6 fast flow sorbents under different temperature and protein loading conditions.

This study has examined the batch binding behaviour of different thermo-responsive co-polymer grafted chromatographic materials under different temperature and protein loading conditions. The effect of molecular composition of poly(N-isopropylacrylamide) (PNIPAAm)-based co-polymers on the phase transition properties has been documented. Sixteen co-polymers of different compositions were synthesized by free radical polymerization methods. Most underwent relatively sharp phase transitions upon application of increasing temperature. However, the value of the lower critical solution temperature (LCST) varied due to differences in co-polymer compositions. In general, it was found that the LCST increased for co-polymers containing more hydrophilic moieties, but decreased for co-polymers with more hydrophobic moieties. Moreover, the LCST increased, together with increased width of the transition temperature, when highly branched monomeric moieties (i.e. N­tert­octyl groups) were present. When bulky side chains (octadecyl or triphenylmethyl groups) were located in the polymer structures the LCST transition was absent. Based on these findings, 6 thermo-responsive co-polymers of different compositions were individually immobilised onto cross-linked Sepharose 6 Fast Flow by a "grafting-from" method. Bovine holo-lactoferrin and bovine holo-transferrin at different concentrations in the range 1-100 mg/mL were then employed as target proteins to evaluate the adsorption behaviour under batch binding conditions with these different polymer grafted Sepharose 6 Fast Flow sorbents at two different temperatures. In general, all sorbents exhibited greater affinity and adsorption capacity for bovine holo-lactoferrin at 50 °C compared to 20 °C. In addition, the affinity and adsorption capacity of bovine holo-lactoferrin with positively charged copolymer grafted Sepharose 6 Fast Flow chromatographic sorbents at 20 °C and 50 °C were much lower than that found for negatively charged copolymer grafted Sepharose 6 Fast Flow sorbents, whilst the opposite trend was found with bovine holo-transferrin due to differences in the surface charge properties of these two proteins, indicative of different separation selectivity. Furthermore, the structure of the side chains present in the grafted copolymer structure was found to affect the adsorption performance of both proteins at 20 °C and 50 °C.

Parallel enrichment of polyphenols and phytosterols from Pinot noir grape seeds with molecularly imprinted polymers and analysis by capillary high-performance liquid chromatography electrospray ionisation tandem mass spectrometry.

This investigation describes an integrated workflow for the parallel extraction and recovery of polyphenols and phytosterols from Pinot noir grape seeds. Using (E)-resveratrol and stigmasterol as exemplars, the approach employs two different molecular imprinted polymers in tandem for the extraction of these compounds and their subsequent analysis by capillary high-performance liquid chromatography (capHPLC) interfaced with electrospray ionisation tandem mass spectrometry (ESI MS/MS). Information on the selectivity of the solid-phase extraction processes was obtained through analysis of the binding behaviour of (E)-resveratrol- and stigmasterol-imprinted polymers using structurally similar polyphenols or phytosterols with the extent of binding determined from the capHPLC-ion trap ESI MS/MS data. This study documents with Pinot noir grape seed extracts and optimised solid-phase extraction protocols that the (E)-resveratrol-templated MIP enabled a very high recovery (99%) of the health-beneficial polyphenol (E)-resveratrol with co-purification of procyanidin and catechin/epicatechin. Further, the stigmasteryl-3-O-methacrylate-templated polymer resulted in high recovery (96%) of the phytosterol stigmasterol with co-purification of campesteryl glycoside. The results also demonstrate that rapid and high-resolution capHPLC-ESI MS/MS methods can be used as part of the work flow for selectivity optimisation and monitoring of the performance of MIPs intended for use in the solid-phase extraction of bioactive molecules with nutraceutical properties from agricultural waste streams.

Aqueous normal-phase retention of nucleotides on silica hydride columns.

The use of silica hydride-based stationary phases for the retention and analysis of nucleotides has been investigated. Both reversed-phase columns with a hydride surface underneath as well as those with an unmodified or a minimally modified hydride material were tested. With these systems, an aqueous normal-phase mode was used with high organic content mobile phases in combination with an additive to control pH for the retention of the hydrophilic nucleotides. Isocratic and gradient elution formats have been used to optimize separations for mixtures containing up to seven components. All conditions developed are suitable for methods that utilize mass spectrometry detection.

Investigations into the interaction thermodynamics of TRAP-related peptides with a temperature-responsive polymer-bonded porous silica stationary phase.

The interaction thermodynamics of the thrombin receptor agonistic peptide (TRAP-1), H-Ser-Phe-Leu-Leu-Arg-Asn-Pro-OH, and a set of alanine scan substitution peptides, have been investigated with an n-octadecylacrylic polymer-bonded porous silica (Sil-ODA18) and water-acetonitrile mobile phases at temperatures ranging from 5 to 80 °C in 5 °C increments. The retention of these peptides on the Sil-ODA18 stationary phase decreased as the water content in the mobile phase was lowered from 80% (v/v) to ca. 45% (v/v) and reached a minimum value for each peptide at a specific water-acetonitrile composition. Further decreases in the water content of the mobile phase led to increased retention. The magnitude of the changes in enthalpy of interaction, Δ H a s s o c 0 , changes in entropy of interaction, Δ S a s s o c 0 , and changes in heat capacity, Δ C p 0 , were found to be dependent on the molecular properties of the mobile phase, the temperature, the structure/mobility of the stationary phase, and the conformation and solvation state of the peptides. With water-rich mobile phases, the retention behaviour of the TRAP analogues was dominated by enthalpic processes, consistent with the participation of strong hydrogen bonding effects, but became dominated by entropic effects with acetonitrile-rich mobile phases as the temperature was increased. These changes in the retention behaviour of these TRAP peptides are consistent with the generation of water or acetonitrile clusters in the mobile phase depending on the volume fractions of the organic solvent as the Sil-ODA18 stationary phase transitions from its crystalline to its isotropic state.