Monolithic Chromatographic Supports Research Articles

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

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.

Monolithic chromatographic supports employed in virus separation and purification

Monolithic chromatographic supports employed in virus separation and purification

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).

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.

Enhanced detection of pathogenic enteric viruses in coastal marine environment by concentration using methacrylate monolithic chromatographic supports paired with quantitative PCR

Currently, around 50% of the world's population lives in towns and cities within 100 km of the coast. Monitoring of viruses that are frequently present in contaminated coastal environments, such as rotavirus (RoV) and norovirus (NoV), which are also the major cause of human viral gastroenteritis, is essential to ensure the safe use of these water bodies. Since exposure to as few as 10–100 particles of RoV or NoV may induce gastrointestinal disease, there is a need to develop a rapid and sensitive diagnostic method for their detection in coastal water samples. In this study, we evaluate the application of methacrylate monolithic chromatographic columns, commercially available as convective interaction media (CIM®), to concentrate pathogenic enteric viruses from saline water samples prior to virus quantification by one-step reverse transcription quantitative PCR (RT-qPCR). Using RoV and NoV as model enteric viruses, we present our results on the most effective viral concentration conditions from saline water matrices using butyl (C4) hydrophobic interaction monolithic support (CIM® C4). C4 monolithic columns exhibit a good capacity to bind both RoV and NoV and both viruses can be eluted in a single step. Our protocol using a 1 ml C4 column enables processing of 400 ml saline water samples in less than 60 min and increases the sensitivity of RoV and NoV detection by approximately 50-fold and 10-fold respectively. The protocol was also scaled up using larger capacity 8 ml C4 columns to process 4000 ml of seawater samples with concentration factors of 300-fold for RoV and 40-fold for NoV, without any significant increase in processing time. Furthermore, C4 monolithic columns were adapted for field use in an on-site application of RoV concentration from seawater samples with performance equivalent to that of the reference laboratory setup. Overall, the results from successful deployment of CIM C4 columns for concentration of rotavirus and norovirus in seawater samples reiterate the utility of monolithic supports as efficient, scalable and modular preparative tools for processing environmental water samples to enhance viral detection using molecular methods.

Titanium dioxide nanoparticle coating of polymethacrylate-based chromatographic monoliths for phosphopetides enrichment

Metal oxide affinity chromatography has been one of the approaches for specific enrichment of phosphopeptides from complex samples, based on specific phosphopeptide adsorption forming bidentate chelates between phosphate anions and the surface of a metal oxide, such as TiO2, ZrO2, Fe2O3, and Al2O3. Due to convective mass transfer, flow-independent resolution and high dynamic binding capacity, monolith chromatographic supports have become important in studies where high resolution and selectivity are required. Here, we report the first synthesis and characterization of immobilisation of rutile TiO2 nanoparticles onto organic monolithic chromatographic support (CIM-OH-TiO2). We demonstrate the specificity of CIM-OH-TiO2 column for enrichment of phosphopeptides by studying chromatographic separation of model phosphorylated and nonphosphorylated peptides as well as proving the phosphopeptide enrichment of digested bovine α-casein. The work described here opens the possibility for a faster, more selective enrichment of phosphopeptides from biological samples that will enable future advances in studying protein phosphorylation.

Enhancing Quantitative‐PCR‐based Detection of Pathogenic Enteric Viruses in Coastal Marine Environment Using Monolithic Chromatographic Supports

Enhancing Quantitative‐PCR‐based Detection of Pathogenic Enteric Viruses in Coastal Marine Environment Using Monolithic Chromatographic Supports

CIM® monolith chromatography-enhanced ELISA detection of proteins in artists' paints: Ovalbumin as a case study

ELISA (Enzyme-linked ImmunoSorbent Assay), as one possible application of antibody-based methods, has proved to be a good candidate for the determination of proteins in works of art due to the low limit of detection, high sensitivity (in the nanogram range), specificity and micro invasiveness of required sampling. Despite the advantages of this method, interference by metal ions present in pigments, lipids, saccharides and in other components of paints, grounds, and varnishes may cause several problems for incontestable identification of proteins. To overcome the drawbacks of ELISA, a novel way to purify the extracted mixtures to predominantly protein fractions, by the DEAE-modified (weak anion exchange) CIM® (Convective Interaction Media) chromatographic monoliths was introduced. Utilisation of monolithic chromatographic supports greatly enhanced the ELISA detection of ovalbumin in the model paint samples (aged and non-aged), for at least one tenfold dilution of a sample. In several cases the purification enabled detection of the target protein, otherwise not possible with non-purified samples. For the first time in the field of Cultural Heritage, the combined approach of ELISA and CIM® monolith-supported extraction was successfully tested also for the detection of protein in real-case paint layers.

A new application of monolithic supports: The separation of viruses from one another

The emergence of next-generation “deep” sequencing has enabled the study of virus populations with much higher resolutions. This new tool increases the possibility of observing mixed infections caused by combinations of plant viruses, which are likely to occur more frequently than previously thought. The biological impact of co-infecting viruses on their host has yet to be determined and fully understood, and the first step towards reaching this goal is the separation and purification of individual species. Ion-exchange monolith chromatography has been used successfully for the purification and concentration of different viruses, and number of them have been separated from plant homogenate or bacterial and eukaryotic lysate. Thus, the question remained as to whether different virus species present in a single sample could be separated. In this study, anion-exchange chromatography using monolithic supports was optimized for fast and efficient partial purification of three model plant viruses: Turnip yellow mosaic virus, Tomato bushy stunt virus, and Tobacco mosaic virus. The virus species, as well as two virus strains, were separated from each other in a single chromatographic experiment from an artificially mixed sample. Based on A260/280 ratios, we were able to attribute specific peaks to a certain viral morphology/structure (icosahedral or rod-shaped). This first separation of individual viruses from an artificially prepared laboratory mixture should encourage new applications of monolithic chromatographic supports in the separation of plant, bacterial, or animal viruses from all kinds of mixed samples.

The Detection Rate of Enteric Viruses and Clostridium difficile in a Waste Water Treatment Plant Effluent.

Waste water treatment plant (WWTP) is considered as an important source of surface water contamination by enteric pathogens. In this study, we describe the occurrence of enteric viruses (group A rotaviruses, noroviruses, astroviruses, sapoviruses, hepatitis A virus, and hepatitis E virus) and Clostridium difficile in the effluent of a wastewater treatment plant during a 1-year period. Enteric viruses were simultaneously and efficiently concentrated in a single step using methacrylate monolithic chromatographic support. Rotaviruses, noroviruses (genogroup I and II), and sapoviruses were detected in all 12 concentrated samples, whereas astroviruses were not detected in August and September and hepatitis A and E viruses were not detected at all. Clostridium difficile was detected in all samples and altogether 121 strains were isolated and grouped into 32 different ribotypes of which 014/020 and 010 were most prevalent. Pathogens detected in WWTP effluent partially reflect the epidemiological situation of enteric viruses and C. difficile in human population and open the discussion on implementation of possible techniques for virus and bacteria removal from WWTP effluent prior to release into the surface water system.

A multimodal histamine ligand for chromatographic purification of plasmid DNA

To exploit different chromatographic modes for efficient plasmid DNA (pDNA) purification a novel monolithic chromatographic support bearing multimodal histamine (HISA) groups was developed and characterized. Electrostatic charge of HISA groups depends on the pH of the mobile phase, being neutral above pH 7 and becoming positively charged below. As a consequence, HISA groups exhibit predominantly ion-exchange character at low pH values, which decreases with titration of the HISA groups resulting in increased hydrophobicity. This feature enabled separation of supercoiled (sc) pDNA from other plasmid isoforms (and other process related impurities) by adjusting salt or pH gradient. The dynamic binding capacity (DBC) for a 5.1kbp large plasmid at pH 5 was 4.0mg/ml under low salt binding conditions, remaining relatively high (3.0mg/ml) even in the presence of 1.0M NaCl due to the multimodal nature of HISA ligand. Only slightly lower DBC (2.7mg/ml) was determined under preferentially hydrophobic conditions in 3.0M (NH4)2SO4, pH 7.4. Open circular and sc pDNA isoforms were baseline separated in descending (NH4)2SO4 gradient. Furthermore, an efficient plasmid DNA separation was possible both on analytical as well as on preparative scale by applying the descending pH gradient at a constant concentration (above 3.0M) of (NH4)2SO4.

Fast purification of the filamentous Potato virus Y using monolithic chromatographic supports

Obtaining pure virus suspensions is an essential step in many applications, such as vaccine production, antibody production, sample preparation for procedures requiring enrichment in viruses and other in vitro characterizations. Purification procedures usually consist of complex, long lasting and tedious protocols involving several ultracentrifugation steps. Such complexity is particularly evident in the case of plant viruses, where the virus needs to be isolated from the complex plant tissue matrix. Convective Interaction Media (CIM) monoliths are chromatographic supports that have been successfully utilized for the purification of large bio-molecules such as viruses, virus like particles and plasmids from various matrixes. In this study a CIM monolith based procedure was developed for the fast purification from plant tissue of the filamentous Potato virus Y (PVY) (virion size, 740nm×11nm), which is one of the most important plant viruses causing great economical losses in potato production. Different mobile phases, chemistries and sample preparation strategies were tested. The presence of the virus in the chromatographic fraction was monitored with viral RNA quantification (RT-qPCR), viral protein purity estimation (SDS-PAGE) and viral particle integrity observation (transmission electron microscopy). The optimized procedure involves initial clarification steps, followed by chromatography using CIM quaternary amine (QA) monolithic disk column. In comparison to classical purification procedure involving ultracentrifugation through sucrose and caesium chloride, the developed CIM-QA purification achieved comparable yield, concentration and purity. Plant nucleic acids were successfully removed. Purification showed good reproducibility and moreover it reduced the purification time from four working days required for classic purification to a day and a half. This is the first study where a filamentous virus was purified using CIM monolithic supports. The advantages of this new purification procedure make it an attractive method in serological diagnostic tool production, which requires purified viruses for the immunization step. Moreover, the outcome of this study could serve as starting point for the improvement of the purification methods of other important filamentous viruses.

New monolithic chromatographic supports for macromolecules immobilization: Challenges and opportunities

This mini-review reports on some recent advances in the field of immobilized protein employing both silica and polymer-based monoliths as supports, and their application in affinity chromatography and immobilized enzyme reactors (IMERs) developments. The major emphasis is put on some interesting challenges and opportunities related to the development of new monolithic affinity supports based on biofriendly sol-gel inorganic monoliths with entrapped proteins and on organic monolithic supports with improved hydrophilicity for IMERs development in proteomic studies. The ease of preparation of monoliths and the multitude of functionalization techniques, make monoliths interesting for an increasing number of biochemical and medical applications.

Peculiarities of a novel bioenzymatic reactor using carbon nanotubes as enzyme activity enhancers: Application to arginase

Multiwalled carbon nanotubes have been entrapped in a porous monolithic chromatographic support. This support was used for the covalent immobilization of the arginase enzyme a novel target in hypertension. The effect of the nanotube (NT) amount into the monolith was analyzed. The obtained results demonstrated the ability of carbon nanotubes to increase significantly the performance of this novel bioactive support.

Concentration and purification of rubella virus using monolithic chromatographic support

The production of economically acceptable viral vaccines of high quality requires simple and efficient methods for purification and concentration of viral particles. Ion-exchange chromatography (IEC) has become one of commonly used methods for large-scale downstream purification of viruses. Viruses possess different biological and/or biochemical properties and therefore IEC conditions must be established specifically for each virus. Live attenuated rubella virus vaccines have been manufactured and successfully used widely to protect people from rubella and congenital rubella syndrome for almost 40 years. The aim of this study was to search for an efficient method for concentration and purification of rubella virus using IEC. The selected operating conditions using quaternary amine monolithic supports enabled highly efficient binding, purification and concentration of rubella virus from complex biological suspension without additional procedures. Eluted viral particles maintained their infectivity and viral recovery was almost 100%. At the same time, viral preparation was successfully depleted from host cell protein and DNA. This work indicates the possibility of using monoliths to improve the rubella virus yields in productions where high virus titers during cultivation can hardly be achieved.

Preparation of pharmaceutical-grade plasmid DNA using methacrylate monolithic columns

Plasmid DNA (pDNA) used in vaccination and gene therapy has to be highly pure and homogenous, which point out necessity to develop efficient, reproducible and scalable downstream process. Convective Interaction Media (CIM) monolithic chromatographic supports being designed for purification of large molecules and nanoparticles seem to be a matrix of choice for pDNA purification. In present work we describe a pDNA purification process designed on two different CIM monolithic columns, based on anion-exchange (AEX) chromatography and hydrophobic interaction chromatography (HIC) chemistry. HIC monolith enabled separation of supercoiled (sc) pDNA from open circular (oc) pDNA, genomic DNA (gDNA) and endotoxins regardless to flow rates in the range at least up to 380 cm/h. Dynamic binding capacity of new HIC monolith is up to 4 mg of pDNA per milliliter of support. Combination of both chromatographic steps using optimized CaCl 2 precipitation enabled production of pure pDNA, satisfying all regulatory requirements. Process was found to be reproducible, scalable, and exhibits high productivity. In addition, in-line monitoring of pDNA purification process is shown, using CIM DEAE disk monolithic columns.

Applications of methacrylate‐based monolithic supports for speciation analysis

Liquid chromatography combined with element specific detection is commonly applied in speciation analysis. In these analyses, to obtain reliable data chemical species should not be transformed. To preserve chemical species during the separation step, fast chromatographic procedures and mild separation conditions are required. Monolithic supports that enable rapid chromatographic separations have rarely been used in speciation analysis. Methacrylate-based anion- and cation-exchange monolithic supports offer separation of charged chemical species of elements and can be used as a complementary tool to particle-packed liquid chromatographic columns. The present paper presents an overview of successful applications of methacrylate-based monolithic supports in speciation of zinc (Zn), chromium (Cr), and aluminium (Al) in environmental, occupational health, and biological samples. Measures of analytical performance of convective interaction media (CIM) monolithic chromatographic supports, namely selectivity, sensitivity, and time of analysis, are compared to those of particle-packed columns. The potential of CIM monolithic chromatography in speciation analysis is critically discussed. Direct comparison of the experimental data in speciation of elements by ion-exchange monolithic and fast protein liquid chromatography is reported for the first time. Finally, some recommendations are given for further investigations of the potential of monolithic chromatography and its implementations in different fields of element speciation analysis.

Separation of hypoviral double-stranded RNA on monolithic chromatographic supports

A procedure based on BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25 mM MOPS, 1.5 M NaCl, 0.1 mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis.

Concentrating rotaviruses from water samples using monolithic chromatographic supports

Rotaviruses are the leading cause of diarrhoea in infants around the globe and, under certain conditions they can be present in drinking water sources and systems. Ingestion of 10–100 viral particles is enough to cause disease, emphasizing the need for sensitive diagnostic methods. In this study we have optimized the concentration of rotavirus particles using methacrylate monolithic chromatographic supports. Different surface chemistries and mobile phases were tested. A strong anion exchanger and phosphate buffer (pH 7) resulted in the highest recoveries after elution of the bound virus with 1 M NaCl. Using this approach, rotavirus particles spiked in 1 l volumes of tap or river water were efficiently concentrated. The developed concentration method in combination with a real time quantitative polymerase chain reaction assay detected rotavirus concentrations as low as 100 rotavirus particles/ml.