Lipase-catalysed changes in essential oils revealed by comprehensive two-dimensional gas chromatography.

Candida antarctica lipase A (CALA) was applied for the chemo-selective enzymatic transesterification of terpene and phenyl alcohols in 35 different essential oil samples. Comprehensive two-dimensional gas chromatography with mass spectrometry (GC×GC‒MS) analysis enabled the separation and tentative identification of a cohort of 125 compounds, allowing the instant visualisation of the reaction process changes, amid the complex chemical background of the samples. The results indicate that 42 out of 79 alcohols so-identified were fully or partially esterified within 48 h of reaction, with primary alcohols being the substrates of preference of the enzyme (90-100% conversion), followed by secondary alcohols (mostly ~ 80-100% conversion). No significant conversion of tertiary alcohols and phenols was observed using the tested conditions. Overall, the enzyme's performance was consistent for primary alcohol substrates identified in multiple samples of different compositions. The observed selectivity, efficiency, robustness, scalability (enzyme/substrate working concentration ratio > 1:160), potential reusability, mild reaction conditions, and other factors make this process a greener and more sustainable alternative for industry applications, particularly for the manufacture of novel flavours and fragrances.

Adsorption of a Humanized Monoclonal Antibody onto Thermoresponsive Copolymer-Grafted Sepharose Fast Flow Sorbents.

The batch adsorption behavior of a humanized monoclonal antibody (hIgG2 mAb) with thermoresponsive polymer (TRP)-modified Sepharose Fast Flow sorbents with different compositions of grafted copolymers is described. At high protein loadings, the adsorption with negatively charged copolymer-modified sorbents exhibited S-shaped isotherms in most cases, indicative of unrestricted multilayer adsorption. The adsorption capacity of the negatively charged copolymer-modified sorbents increased with an increase in the applied environmental temperature due to increased protein-sorbent surface hydrophobic and electrostatic interactions. The affinity of the hIgG2 mAb for a positively charged copolymer-grafted sorbent was much lower than that found for the negatively charged copolymer-grafted sorbents at both 20 and 50 °C due to electrostatic repulsive effects. This study has documented that the molecular functionalities of the grafted copolymer can significantly affect the adsorption behavior of this humanized mAb at both 20 and 50 °C with the isothermal dependencies revealing subtle effects due to copolymer composition.

Isothermal modelling of protein adsorption to thermo-responsive polymer grafted Sepharose Fast Flow sorbents.

In this study, five adsorption isotherm models, that is, the Langmuir, Freundlich, Langmuir-Freundlich, Temkin and Brunauer-Emmett-Teller isotherms, were utilized for the analysis of the experimental adsorption data for six classes of poly(N-isopropylacrylamide)-based thermo-responsive copolymer-grafted Sepharose Fast Flow sorbents of different copolymer compositions with two structurally related proteins, namely bovine holo-lactoferrin and bovine holo-transferrin at 20 and 50°C. The experimental data for bovine holo-lactoferrin could be mathematically fitted to the Freundlich and Temkin isotherms when the protein feed concentrations were in the range of 1-40 mg/mL at both 20 and 50°C. Similar analysis of the binding of the homologous protein, bovine holo-transferrin, to the same thermo-responsive copolymer-grafted sorbents revealed that the experimental data could be fitted to the Langmuir, Freundlich and Temkin isotherms with coefficients of determination value over 0.90.

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.

Interactions between an amphipathic di-histidine peptide and a metal affinity chromatographic resin derived from a bis(tacn)butane chelating ligand.

The interactive behavior of an amphipathic peptide with the Cu2+ , Ni2+ , and Zn2+ complexes of 1,4-bis(triazacyclonon-1-yl)butane), bis(tacn)but , immobilized onto Sepharose CL-4B, has been investigated. The effects of incubation time, as well as the incubation buffer pH and ionic strength, have been examined. The binding data have been interrogated using Langmuir, Langmuir-Freundlich, bi-Langmuir, and Temkin isothermal models and Scatchard plots. These results confirm that this amphipathic peptide binds with relatively high capacities to the immobilized Cu2+ - and Ni2+ -1,4-bis(triazacyclonon-1-yl)butane)-Sepharose CL-4B sorbents via at least two discrete sites. However, the corresponding immobilized Zn2+ -sorbent had low binding capacity. Moreover, the magnitude of the binding capacities of these sorbents was dependent on the pH and ionic strength of the incubation buffer. These results are relevant to the isolation of E. coli expressed recombinant proteins that incorporate this and related amphipathic peptide tags, containing two or more histidine residues, located at the N- or C-terminus of the recombinant protein, and the co-purification of low abundance host cell proteins of diverse structure, by immobilized metal ion affinity chromatographic methods.

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.

Poly(N-4-vinylbenzyl-1,4,7-triazacyclononane) Copper Complex Grafted Solid Catalyst for Oxidative Polymerization of 2,6-Dimethylphenol.

A new solid phase catalyst, poly(N-4-vinylbenzyl-1,4,7-triazacyclononane) copper(I) complex, grafted onto polystyrene particles, has been employed for the oxidative polymerization of 2,6-dimethylphenol using an aqueous biphasic (water/toluene) solvent system. The solid catalyst was synthesized by first grafting N-(4-vinylbenzyl)-1,4,7-triaza-cyclononane onto polystyrene particles using a radical mediated polymerization method and next by creating the polymer-metal complex of copper-triazacyclononane with these modified particles. Poly(2,6-dimethyl-1,4-phenylene oxide) was successfully obtained from the polymerization of 2,6-dimethylphenol using this new metal-organic solid phase catalyst.

N-terminal processing of affinity-tagged recombinant proteins purified by IMAC procedures.

The ability of a new class of metal binding tags to facilitate the purification of recombinant proteins, exemplified by the tagged glutathione S-transferase and human growth hormone, from Escherichia coli fermentation broths and lysates has been further investigated. These histidine-containing tags exhibit high affinity for borderline metal ions chelated to the immobilised ligand, 1,4,7-triazacyclononane (tacn). The use of this tag-tacn immobilised metal ion affinity chromatography (IMAC) system engenders high selectivity with regard to host cell protein removal and permits facile tag removal from the E. coli-expressed recombinant protein. In particular, these tags were specifically designed to enable their efficient removal by the dipeptidyl aminopeptidase 1 (DAP-1), thus capturing the advantages of high substrate specificity and rates of cleavage. MALDI-TOF MS analysis of the cleaved products from the DAP-1 digestion of the recombinant N-terminally tagged proteins confirmed the complete removal of the tag within 4-12 h under mild experimental conditions. Overall, this study demonstrates that the use of tags specifically designed to target tacn-based IMAC resins offers a comprehensive and flexible approach for the purification of E. coli-expressed recombinant proteins, where complete removal of the tag is an essential prerequisite for subsequent application of the purified native proteins in studies aimed at delineating the molecular and cellular basis of specific biological processes.

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.

Recent Developments in Chemical Synthesis with Biocatalysts in Ionic Liquids.

Over the past decade, a variety of ionic liquids have emerged as greener solvents for use in the chemical manufacturing industries. Their unique properties have attracted the interest of chemists worldwide to employ them as replacement for conventional solvents in a diverse range of chemical transformations including biotransformations. Biocatalysts are often regarded as green catalysts compared to conventional chemical catalysts in organic synthesis owing to their properties of low toxicity, biodegradability, excellent selectivity and good catalytic performance under mild reaction conditions. Similarly, a selected number of specific ionic liquids can be considered as greener solvents superior to organic solvents owing to their negligible vapor pressure, low flammability, low toxicity and ability to dissolve a wide range of organic and biological substances, including proteins. A combination of biocatalysts and ionic liquids thus appears to be a logical and promising opportunity for industrial use as an alternative to conventional organic chemistry processes employing organic solvents. This article provides an overview of recent developments in this field with special emphasis on the application of more sustainable enzyme-catalyzed reactions and separation processes employing ionic liquids, driven by advances in fundamental knowledge, process optimization and industrial deployment.

N-Heterocyclic-based adsorbents for antibody purification-effect of ligand structure.

A new set of ligands based on substituted pyridine and other N-heterocyclic structures, possessing an aliphatic primary amino group tether and an exocyclic sulphur atom, has been prepared and immobilized onto epoxy-activated matrices such as Sepharose 6 Fast Flow®. The derived adsorbents have been evaluated for their utility to capture and purify humanized monoclonal antibodies. Favourable binding properties were assessed from screening assays to determine optimal conditions for the capture and elution of the monoclonal antibodies. Static and dynamic binding experiments were employed to derive the equilibrium dissociation constants KD 's and binding capacities Qmax 's. Typically, the KD values were in the range of 2-5 µM and the Qmax values between 20 and 75 mg mAb/ml resin, depending on the stereo-electronic properties of the substituent in the N-heterocyclic ring structure. The effect of ligand structure on the selectivity of these adsorbents was also investigated, and criteria for their use in the purification of monoclonal antibodies from cell culture supernatants established.

Application of 4'-terpyridinylsulfanylethylamine resins for the purification of monoclonal antibodies by mixed-mode chromatography.

In this study, a pyridine-based compound, 4'-terpyridinylsulfanylethylamine (4'-TerPSEA), has been employed as a ligand to purify via mixed-mode chromatographic procedures a humanised monoclonal antibody of the IgG1 sub-class directly from crude supernatants derived from cultured CHO cells. The antibody binding capacity, selectivity and reusability of the adsorbent, derived from the immobilisation of this ligand onto Sepharose FF™, were compared to a Protein A affinity resin. The chromatographic performance of this mixed mode adsorbent was similar to that shown by the Protein A-based adsorbent with this IgG1 mAb. In addition, the IgG1 mAb was able to bind to the immobilised 4'-TerPSEA under reducing conditions. Through the use of papain-digested IgG1 mAb, fractionated with both the 4'-TerPSEA and Protein A adsorbents, it was found that this IgG1 mAb preferentially bound to the immobilised 4'-TerPSEA Sepharose FF™ resin through its Fc region.

Purification of a recombinant human growth hormone by an integrated IMAC procedure.

In this study, integration of three discrete process aspects of the IMAC purification of Escherichia coli expressed recombinant proteins has been investigated. To this end, novel N-terminally tagged human growth hormone variants (tagged-vhGHs) have been expressed in E. coli by tank fermentation and captured directly from the cell lysate by a new IMAC approach. The chelating ligands used were 1,4,7-triaza-cyclononane (tacn) and bis(1,4,7-triazacyclononyl)-propane (dtnp) with copper(II) as the immobilised metal ion. The N-terminal tags were specifically selected for their potential to bind to these immobilised complexes and also for their ease of removal from the tagged protein by the dipeptidyl peptidase, DAP-1. Low levels of detergents in the binding buffer did not dramatically affect the purification, but increased concentrations of NaCl in the loading buffer improved the binding performance. The same IMAC systems, operated in the 'negative' adsorption chromatographic mode, could be used to obtain the purified mature human growth hormone variant, as assessed by MALDI-TOF and N-terminal sequencing studies, following removal of the affinity tag by the dipeptidyl peptidase 1. Western immunoblot analysis of the eluted fractions of both the tagged and de-tagged vhGH demonstrated significant clearance of E. coli host cell proteins (HCPs). Further, these IMAC resins can be used multiple times without the need for metal ion re-charging between runs. This study thus documents an integrated approach for the purification of specifically tagged recombinant proteins expressed in genetically modified E. coli.

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.