Monolithic Supports Research Articles

Enterokinase monolithic bioreactor as an efficient tool for biopharmaceuticals preparation: on-line cleavage of fusion proteins and analytical characterization of released products

One of the most popular enzymes used for the in vitro cleavage of fusion proteins is enterokinase (EK, E.C. 3.4.21.9). EK cleaves with high specificity after the sequence Asp4-Lys (DDDDK), which allows the fusion protein to preserve its native amino acid terminus without any additional unwanted cleavage residue from the recognition sequence. However, the complete removal of EK after protein cleavage is a critical step to ensure protein identity and stability.As enzyme immobilization increases stability and reusability of the biocatalyst while reducing operating costs and sample contamination, in this work we report the covalent immobilization of recombinant EK (rEK) on monolithic chromatographic supports with different binding chemistries for the development of a rEK-chromatographic-bioreactor. An on-line assay for the determination of the activity of the immobilized rEK was set up using a synthetic substrate (Gly-Asp4-Lys-β-naphthylamide, GD4K-NA). The assay was used to study the improvement of the operational conditions (temperature and flow rate) on hydrolytic activity of the bioreactor. The immobilization yields, as well as the cleavage activity of immobilized rEK on GD4K-NA, were highly satisfactory when the immobilized enzyme reactor was used in recirculation. The ability of the immobilized rEK to cleave fusion proteins was tested by recirculation of thioredoxin (Trx)-TB10.4 and Trx-Ag85B His-tagged proteins yielding the mature antigens TB10.4 and Ag85B, to be used in the preparation of potential novel glycovaccines against tuberculosis. The prepared rEK-based immobilized enzyme reactors proved to efficiently cleave the considered fusion proteins even if the cleavage specificity at the canonical site was not fully achieved. The immobilized rEK showed very good stability and reusability.

New stationary phase for capillary liquid chromatography based on polydimethylsiloxane immobilized onto a Monolithic Silica Support

AbstractA new stationary phase based on polydimethylsiloxane immobilized onto a monolithic silica support was prepared for performing reversed‐phase capillary liquid chromatography. Silica monoliths, using tetramethoxysilane as precursor, and hybrid monoliths, using tetramethoxysilane and methyltrimethoxysilane as precursors, were prepared by the sol–gel process. Polydimethylsiloxane was deposited on the monolithic matrix and its immobilization was performed through thermal treatment. Characterization was made by field‐emission scanning electron microscopy and by chromatographic evaluation for the separation of six alkylbenzenes. An efficiency of 79 500 plates/m was reached for columns of 200 μm id, demonstrating that it would be a promising alternative to a reversed‐phase column in capillary liquid chromatography.

Inside Front Cover: New stationary phase for capillary liquid chromatography based on polydimethylsiloxane immobilized onto a Monolithic Silica Support

Inside Front Cover: New stationary phase for capillary liquid chromatography based on polydimethylsiloxane immobilized onto a Monolithic Silica Support

Arginine homopeptides for plasmid DNA purification using monolithic supports

Purification of plasmid DNA targeting therapeutic applications still presents many challenges, namely on supports and specific ligand development. Monolithic supports have emerged as interesting approaches for purifying pDNA due to its excellent mass transfer properties and higher binding capacity values. Moreover, arginine ligands were already described to establish specific and preferential interactions with pDNA. Additionally, some studies revealed the ability of arginine based cationic peptides to condense plasmid DNA, which increased lengthening can result in strongest interactions with higher binding capacities for chromatographic purposes of large molecules such as pDNA. In this work, arginine homopeptides were immobilized in monolithic supports and their performance was evaluated and compared with a single arginine monolithic column regarding supercoiled (sc) plasmid DNA purification. Specific interactions of arginine based peptides with several nucleic acids present in a clarified Escherichia coli lysate sample showed potential for the sc pDNA purification. Effectively, the immobilization of the arginine homopeptides became more functional compared with the single arginine amino acid, showing higher binding capacities, which was also reflected in the intensity of the interactions. The combination of structural versatilities of monoliths with the specificity of arginine peptides raised as a promising strategy for sc pDNA purification.

Delivery of Brain-Derived Neurotrophic Factor by 3D Biocompatible Polymeric Scaffolds for Neural Tissue Engineering and Neuronal Regeneration.

Biopolymers are increasingly employed for neuroscience applications as scaffolds to drive and promote neural regrowth, thanks to their ability to mediate the upload and subsequent release of active molecules and drugs. Synthetic degradable polymers are characterized by different responses ranging from tunable distension or shrinkage to total dissolution, depending on the function they are designed for. In this paper we present a biocompatible microfabricated poly-ε-caprolactone (PCL) scaffold for primary neuron growth and maturation that has been optimized for the in vitro controlled release of brain-derived neurotrophic factor (BDNF). We demonstrate that the designed morphology confers to these devices an enhanced drug delivery capability with respect to monolithic unstructured supports. After incubation with BDNF, micropillared PCL devices progressively release the neurotrophin over 21days in vitro. Moreover, the bioactivity of released BDNF is confirmed using primary neuronal cultures, where it mediates a consistent activation of BDNF signaling cascades, increased synaptic density, and neuronal survival. These results provide the proof-of-principle on the fabrication process of micropatterned PCL devices, which represent a promising therapeutic option to enhance neuronal regeneration after lesion and for neural tissue engineering and prosthetics.

Surveillance of human enteric viruses in coastal waters using concentration with methacrylate monolithic supports prior to detection by RT-qPCR

This is the first surveillance study using methacrylate monolithic supports to concentrate environmental coastal water samples, prior to molecular target detection by RT-qPCR. Rotaviruses (RoV) and Noroviruses (NoV) were monitored in a polluted area at the Bay of Koper (Gulf of Trieste, Northern Adriatic Sea) and at a nearby bathing area and mussel farm areas.RoV and NoV are released into the Bay of Koper, with higher rates close to the discharge of the wastewater treatment plant, however, they can be detected at recreational and mussel farming areas. Our results showed that water bodies considered safe based on FC concentrations, can still have low, yet potentially infective, concentrations of human viruses.

АНАЛИЗ НАПРЯЖЕННО-ДЕФОРМИРОВАННОГО СОСТОЯНИЯ МОНОЛИТНОЙ БЕТОННОЙ КРЕПИ ПОДЗЕМНЫХ СООРУЖЕНИЙ НЕКРУГОВОГО ОЧЕРТАНИЯ

Приведены результаты исследований напряженно-деформированного состояния (НДС) крепи и грунтового массива подземных сооружений некругового очертания глубиной заложения до 50 м в неустойчивых наносных породах с заходками от 0,3 до 0,7 м. Исследование выполнено путём моделирования НДС массива грунта и твердеющего бетона монолитной крепи, возведенной вслед за подвиганием забоя.Для реализации предлагаемых технологических решений обеспечивающих крепление подземных сооружений монолитным бетоном для исключения применения временной крепи требуется использование особобыстротвердеющих составов. На основании исследований ряда модификаторов установлены изменения механических свойств наиболее эффективных составов, которые использованы для моделирования бетонной крепи сооружений с изменяющимися во времени характеристиками.Цель: обоснование ресурсосберегающей технологии строительства подземных сооружений некругового очертания в грунтовом массиве.Метод или методология проведения работы: исследования проведены методом конечных элементов (МКЭ) с использованием оптимального планирования эксперимента.Результаты: получены распределения напряжений в твердеющей бетонной крепи сооружений, установлены зоны концентрации напряжений, установлены величины смещений в грунтовом массиве и их распространение на поверхности.Область применения результатов: результаты исследований могут применяться при проектировании вариантов строительства подземных сооружений.

Cu-ZSM-5 zeolite supported on SiC monolith with enhanced catalytic activity for NH3-SCR

Here, we developed a new kind of hetero-structural catalyst through the homogeneous growth of Cu-ZSM-5 zeolite onto silicon carbide (SiC) monolith by using a one-pot hydrothermal method combined with an ion-exchange process in a rotary oven. The new catalyst formulation was tested towards NH3-SCR, where the catalytic activity and stability of Cu-ZSM-5/SiC at T > 350 °C appear significantly higher than those obtained with Cu-ZSM-5 alone. In particular, the NO consumption rate of the former is 7.5 times larger on the average than that of the latter. This remarkable enhancement of catalytic activity is ascribed to the very good thermal conductivity of the SiC monolithic support. This research work could provide the means for the future design of more active and stable monolithic Cu-ZSM5-based catalytic systems with even lower Cu loading than the ones used in present de-NOx applications.

Monolithic chromatographic supports employed in virus separation and purification

Monolithic chromatographic supports employed in virus separation and purification

Poly(glyceryl monomethacrylate-co-ethylene glycol dimethacrylate) monolithic columns with incorporated bare and surface modified gluconamide fumed silica nanoparticles for hydrophilic interaction capillary electrochromatography

This research article presents the preparation and characterization of monolithic capillary columns with incorporated bare fumed silica nanoparticles (FSNPs) and surface coated gluconamide FSNPs and their subsequent use in hydrophilic interaction capillary electrochromatography (HI-CEC) of small relatively polar solutes. The monolithic support was based on the in situ polymerization of glyceryl monomethacrylate (GMM) and ethylene glycol dimethacrylate (EDMA) yielding the poly(GMM-co-EDMA) monolith for the incorporation of bare and gluconamide-FNSPs. The poly(GMM-co-EDMA) monolith functioned as a true “support” for both types of polar FSNPs “stationary phases”. In other words, monolithic capillary columns with “FSNPs stationary phases” were obtained in the sense that the contribution of the monolith proper to solute’ retention was at its minimum. The gluconamide-FSNPs were obtained by reacting the FSNPs with the polar organosilane N-(3-triethoxysilylpropyl)gluconamide either by a pre- or on-column approach yielding p-gluconamide-FSNPs or o-gluconamide-FSNPs, respectively. While the p-gluconamide-FSNPs was coated by an oligosiloxane gluconamide layer as revealed by thermogravimetric analysis, the o-gluconamide-FSNPs are thought to be covered with a monomeric layer of gluconamide ligands as was manifested by the higher plate number obtained on the latter than on the former gluconamide-FSNPs incorporated monolithic columns. In the on-column modification process of FSNPs, the reaction was performed in a closed system whereby atmospheric water vapor are not available to cause the polymerization of the trifunctional organosilane N-(3-triethoxysilylpropyl)gluconamide. Also, the fact that the o-gluconamide-FSNPs incorporated monoliths were made from bare-FSNPs incorporated monoliths may indicate that the bare FSNPs were better dispersed into the monolithic matrix than the p-gluconamide-FSNPs, a condition that might have further contributed to the lower plate count obtained on p-gluconamide- than o-gluconamide-FSNPs incorporated monolithic columns. Overall, o-gluconamide-FSNPs stationary phases and to a lesser extent bare-FSNPs stationary phases proved useful in HI-CEC of small polar solutes, including DMF, formamide, thiourea, some phenols and nucleobases.

OXIDATION OF ETHYL ACETATE ON Fe-Mn OXIDES FROM NITRATES DEPOSITED BY IMPREGNATION ON MONOLITHS OF AISI 304 STAINLESS STEEL

The iron-manganese mixed monolithic catalysts were obtained by impregnation from nitrates as precursors. A monolithic support was studied with and without alumina as surface generator. The characterization consisted in XRD, SEM, Adherence test, SBET and TPR. The monolith was evaluated in the combustion of ethyl acetate. The preparation method was very satisfactory in terms of reproducibility and adherence of the deposited phase. The higher activity of IFeMnx with respect to Al3FeMnx may be attributed for the formation of the solid solution where the Fe is introduced to the bixbyite structure. However, the presence of defective MnOx species in monoliths without alumina which might help to improve their performance is observed.

Back Cover: Application of monolithic chromatographic supports in virus research

Electrophoresis 2017, 38, 2827–2836. DOI: 10.1002/elps.201700152 The cover picture shows the first chromatographic separation of mixed viruses (Virus 1, 2, 3) and virus strains (Virus 2a, 2b) from each other by using methacrylate monoliths and weak anion exchanger, as described in the review “Application of monolithic chromatographic supports in virus research” by Krajacic et al. According to A260/280 ratios (full and dashed line, respectively), it is possible to attribute specific peaks to a certain viral morphology/structure, as demonstrated for icosahedral Tomato bushy stunt and Turnip yellow mosaic virus (Virus 1, 2), or rod-shaped, helically structured Tobacco mosaic virus (Virus 3).

Use of monolithic supports for high-throughput protein and peptide separation in proteomics.

The exclusive properties of monolithic supports enable fast mass transfer, high porosity, low back pressure, easy preparation process and miniaturisation, and the availability of different chemistries make them particularly suitable materials for high-throughput (HTP) protein and peptide separation. In this review recent advances in monolith-based chromatographic supports for HTP screening of protein and peptide samples are presented and their application in HTP sample preparation (separation, enrichment, depletion, proteolytic digestion) for HTP proteomics is discussed. Development and applications of different monolithic capillary columns in HTP MS-based bottom-up and top-down proteomics are overviewed. By discussing the chromatographic conditions and the mass spectrometric data acquisition conditions an attempt is made to present currently demonstrated capacities of monolithic capillary columns for HTP identification and quantification of proteins and peptides from complex biological samples by MS-based proteomics. Some recent advances in basic monolith technology of importance for proteomics are also discussed.

Polymethacrylate-based monoliths as stationary phases for separation of biopolymers and immobilization of enzymes.

The experiences in the production and application of polymethacrylate-based monolithic supports, since their development almost thirty years ago, are presented. The main driving force for the development of new chromatographic supports was the necessity for the isolation and separation of physiologically active biopolymers and their use for therapeutic purposes. For this sake, a development of a method for fast separation, preventing denaturation and preserving their biological activity was necessary. Development of polysaccharide-based supports, followed by the introduction of polymer-based chromatographic media, is shortly described. This development was followed by the advances in monolithic media that are now used for both large- and small-scale separation of biopolymers and nanoparticles. Finally, a short overview is given about the applications of monoliths for sample displacement chromatography, resulting in isolation of physiologically active biomolecules, such as proteins, protein complexes, and nucleic acid, as well as high-throughput sample preparation for proteomic investigations.

Photocatalytic carbon dioxide reduction to fuels in continuous flow monolith photoreactor using montmorillonite dispersed Fe/TiO2 nanocatalyst

Photocatalytic conversion of CO2 to fuels has attracted immense attention because it offers a cleaner energy technology and safer environment. In this study, performance of structured montmorillonite (MMT) dispersed Fe-doped titanium dioxide (Fe/TiO2) nanocomposite was tested for dynamic photo-induced CO2 reduction by H2 to fuels. Cordierite monolithic support was employed in order to improve the photo-activity and reusability of Fe-MMT/TiO2 nanocomposite in a CO2 utilization process. MMT-clay supported Fe/TiO2 samples were prepared by a controlled and direct sol-gel method and were dip-coated over the monolith micro-channels. The efficiency of Fe-loaded MMT/TiO2 for CO2 reduction by H2 toward CO was investigated using a cell type and a monolith photo-reactor under UV-light. The maximum CO yield over 3 wt % Fe-10 wt % MMT-loaded TiO2 catalyst reached 166 μmole g-catal.−1 h−1 at selectivity 99.70%, considerably higher than the amount of CO produced over the MMT/TiO2 (16 μmole g-catal.−1 h−1) and the pure TiO2 (5 μmole g-catal.−1 h−1) catalysts. The other products observed with adequate amounts were CH4 and C2H6. More importantly, photo-activity and stability of Fe-MMT/TiO2 catalyst for CO evolution was significantly improved using monolith photo-reactor compared to the cell type reactor under the same operating conditions. This enactment was evidently due to higher quantum efficiency of monolith photoreactor, improved adsorption-desorption process in a catalyst coated monolith channels and hindered charges recombination by Fe. The reusability of catalyst loaded over the monolithic support showed greater recycling capability than the catalyst dispersed in a cell reactor. This development confirmed higher photo-activity of Fe-MMT/TiO2 photo-catalyst loaded over monolithic support for CO2 photo-reduction to cleaner fuels.

Affinity chromatography on monolithic supports for simultaneous and high-throughput isolation of immunoglobulins from human serum.

Posttranslational modifications of immunoglobulins have been a topic of great interest and have been repeatedly reported as a major factor in disease pathology. Cost-effective, reproducible, and high-throughput (HTP) isolation of immunoglobulins from human serum is vital for studying the changes in protein structure and the following understanding of disease development. Although there are many methods for the isolation of specific immunoglobulin classes, only a few of them are applicable for isolation of all subtypes and variants. Here, we present the development of a scheme for fast and simultaneous affinity purification of α (A), γ (G), and μ (M) immunoglobulins from human serum through affinity monolith chromatography. Affinity-based monolithic columns with immobilized protein A, G, or L were used for antibody isolation. Monolithic stationary phases have a high surface accessibility of binding sites, large flow-through channels, and can be operated at high flow rates, making them the ideal supports for HTP isolation of biopolymers. The presented method can be used for HTP screening of human serum in order to simultaneously isolate all three above-mentioned immunoglobulins and determine their concentration and changes in their glycosylation pattern as potential prognostic and diagnostic disease biomarkers.

Synergistic effect in MMT-dispersed Au/TiO2 monolithic nanocatalyst for plasmon-absorption and metallic interband transitions dynamic CO2 photo-reduction to CO

Structured montmorillonite (MMT) dispersed Au/TiO2 nanocomposite has been designed and synthesized through a facile sol-gel method. Cordierite monolithic support was employed in order to load the catalyst for improved photo-activity and reusability in CO2 utilization process. The samples were characterized by XRD, Raman, SEM, TEM, FTIR, XPS, N2 adsorption-desorption, UV–vis and PL spectroscopy. The synergistic effect of MMT-dispersed Au/TiO2 nanocatalyst was evaluated in a gas-phase dynamic monolith photoreactor system using UV and visible light irradiations. The maximum CO yield over 0.5wt.% Au–10wt.% MMT-loaded TiO2 catalyst reached to 1223μmole g-catal.−1h−1, a 24 fold higher than the amount of CO produced over the 10wt.% MMT/TiO2 and 68 times the amount of CO produced over the bare TiO2 catalyst. The other products observed with considerable amounts were CH4 and C2H6. This enactment under UV-light was due to interband transition of Au in catalyst composite. Enhanced photo-activity under simulated solar energy for CO2-to-CO reduction was due to LSPR effect of Au in the MMT/TiO2 sample. More importantly, the performance of Au-MM/TiO2 catalyst for CO evolution under UV-light was 6 folds higher than using visible light. The synergistic effect between MMT transition metals and Au ions and faster adsorption-desorption process contributed to remarkably enhance dynamic CO2 reduction to CO. The present design of catalyst provides prolonged stability to catalyst while CO evolution sustained even after 44h of operation time. The reaction mechanism developed to understand the role of Au/MMT and monolithic support on the photo-activity and reusability of catalyst for CO2 photo-reduction to fuels.

Semi-high-throughput isolation and N-glycan analysis of human fibrinogen using monolithic supports bearing monoclonal anti-human fibrinogen antibodies.

Fibrinogen (FIB) is a secretory glycoprotein synthesized by hepatocytes that has a key role in blood clotting. Its glycosylation has not been studied in detail and little is known about the biological variability of FIB N-glycosylation, mainly due to the lack of fast, simple, and robust approaches to purify FIB from blood plasma samples. In recent years, customised chromatographic monoliths have been used for a variety of biological applications due to their unique characteristics. Here we describe development and optimisation of monolithic supports bearing monoclonal anti-human fibrinogen antibodies in a single column as well as in multi-well plate formats with high FIB specificity and binding capacity for fast immunoaffinity purification of FIB from human blood samples. The developed semi-high-throughput workflow has been successfully applied for FIB immunoaffinity isolation and subsequent ultra performance liquid chromatography N-glycosylation analysis in ten healthy human individuals, demonstrating the potential of monolithic supports in glycomics studies.

Application of monolithic chromatographic supports in virus research.

Key properties of monolithic chromatographic supports, make them suitable for separation and/or concentration of large biomolecules, especially virus particles and viral genomes. One by one, the studies that have been completed so far, contributed to the knowledge that monolith chromatography has hardly any limitation to be applied in virus research. Viruses of different sizes, possessing icosahedral structure and symmetrical morphology, as well as rod-shaped or filamentous viruses with helical structure, even enveloped ones, all of them could be successfully managed by means of monolith chromatography. Same is true for viral genomes, primarily when being distinct from other nucleic acid forms present in a host cell. This review is exclusively focused on viruses. It describes the application of monolith chromatography to different problematics within the virus research field. The reviewed achievements offer new possibilities and trigger new aspects in virology.

Photo-induced CO2 reduction by hydrogen for selective CO evolution in a dynamic monolith photoreactor loaded with Ag-modified TiO2 nanocatalyst

Ag-promoted TiO2 nanoparticles immobilized over the cordierite monolithic support for dynamic and selective photo-reduction of CO2 to CO by the use of hydrogen has been investigated. Ag-loaded TiO2 NPs synthesized by a facile sol–gel method were coated over the monolith channels by dip-coating method. The samples were characterized by XRD, Raman, FTIR, SEM, TEM, XPS, N2 adsorption–desorption, UV–Vis and PL spectroscopy. The photo-activity test of Ag-modified TiO2 NPs was conducted for dynamic photocatalytic CO2 reduction with H2 as a reductant via a reverse water gas shift (RWGS) reaction in a cell type and monolith photo-reactors. Using 5 wt. % Ag/TO2 NPs, CO2 was energetically converted to CO with a yield rate 1335 μmole g-catal.−1 h−1, a 111 fold-higher than the amount of CO produced over the pure TiO2 catalyst. More importantly, photo-activity of Ag/TiO2 catalyst for CO evolution can be improved by 209 fold using monolith photo-reactor than the cell type reactor under the same operating conditions. This enactment was evidently due to the efficient light harvesting with larger illuminated surface area inside monolith micro-channels and efficient charges separation in the presence of Ag-metal. The reusability of Ag/TiO2 NPs loaded over the monolithic support showed favorable recycling capability than the catalyst dispersed in a cell reactor. A possible reaction mechanism for this observation has been discussed in detail.