Convective Interaction Media Research Articles

Purification of the Staphylococcus aureus bacteriophages VDX-10 on methacrylate monoliths

Bacteriophages (phages) are known to be useful in many fields from medicine to agriculture, and for a broad range of applications, including phage therapy and phage display. For some applications, especially in medicine, high purity and viability of phages are required. Methacrylate monoliths (Convective Interaction Media [CIM] monolithic columns), designed for purification of bionanoparticles, were applied for the purification of Staphylococcus aureus phages VDX-10 from bacterial lysate. With a single step purification method, more than 99% of host cell DNA and more than 90% of proteins were removed, with 60% recovery of viable phages. Comparable results were obtained when the purification method was scaled-up from a CIM monolithic disk to a larger CIM monolithic column. Additionally, the dynamic binding capacity of a methacrylate monolith column for S. aureus phages VDX-10 was determined.

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.

Bioaffinity chromatography on monolithic supports

Affinity chromatography on monolithic supports is a powerful analytical chemical platform because it allows for fast analyses, small sample volumes, strong enrichment of trace biomarkers and applications in microchips. In this review, the recent research using monolithic materials in the field of bioaffinity chromatography (including immunochromatography) is summarized and discussed. After giving an introduction into affinity chromatography, information on different biomolecules (antibodies, enzymes, lectins, aptamers) that can act as ligands in bioaffinity chromatography is presented. Subsequently, the history of monoliths, their advantages, preparation and formats (disks, capillaries and microchips) as well as ligand immobilization techniques are mentioned. Finally, analytical and preparative applications of bioaffinity chromatography on monoliths are presented. During the last four years 37 papers appeared. Protein A and G are still most often used as ligands for the enrichment of immunoglobulins. Antibodies and lectins remain popular for the analysis of mainly smaller molecules and saccharides, respectively. The highly porous cryogels modified with ligands are applied for the sorting of different cells or bacteria. New is the application of aptamers and phages as ligands on monoliths. Convective interaction media (epoxy CIM disks) are currently the most used format in monolithic bioaffinity chromatography.

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.

A novel method for concentrating hepatitis A virus and caliciviruses from bottled water

Human enteric viruses are detected frequently in various types of environmental water samples, such as irrigation water, wastewater, recreational water, ground or subsurface water and even drinking water, constituting a primary source of gastroenteritis or hepatitis outbreaks. Only a few, but still infective number of viral particles are normally present in water samples, therefore an efficient virus concentration procedure is essential prior to molecular detection of the viral nucleic acid. In this study, a novel chromatographic technology, Convective Interaction Media ® (CIM) monolithic supports, were optimized and applied to the concentration of hepatitis A virus (HAV) and feline calicivirus (FCV), a surrogate of norovirus (NoV), from water samples. Two-step real-time RT-qPCR was used for quantitation of the virus concentration in the chromatographic fractions. Positively charged CIM QA (quaternary amine) monolithic columns were used for binding of HAV and FCV present in previously inoculated 1.5 l bottled water samples. Column bound viruses were eluted from the monolith using 1 M NaCl to a final volume of 15 ml. Elution volume was concentrated further by ultracentrifugation. When the CIM/ultracentrifugation method was compared with another concentration method employing positively charged membranes and ultrafiltration, the recovery of HAV was improved by approximately 20%.

Convective Interaction Media Monolithic Chromatography with ICPMS and Ultraperformance Liquid Chromatography−Electrospray Ionization MS Detection: A Powerful Tool for Speciation of Aluminum in Human Serum at Normal Concentration Levels

A new analytical procedure using separation support based on Convective Interaction Media (CIM) was developed for speciation of Al in human serum at normal concentration levels. The separation of proteins was performed on a weak anion exchange CIM diethylamine monolithic column. Isocratic elution with buffer A (0.05 M tris(hydroxymethyl)aminomethane-hydrochloric acid + 0.03 M sodium hydrogen carbonate) was applied for 5 min, followed by linear gradient elution from 100% buffer A to 100% buffer B (buffer A + 1 M ammonium chloride) for the next 40 min. Separation of proteins was followed by UV detection at 278 nm. Separated Al species were detected online by inductively coupled plasma mass spectrometry. It was experimentally proven that 91 +/- 7% of Al in human serum was eluted under the transferrin peak. Transferrin was identified on the basis of the retention volume and by ACQUITY ultraperformance liquid chromatography-electrospray ionization mass spectrometry. The problem of extraneous contamination with Al was successfully overcome by using efficient cleaning procedures of eluents and chromatographic supports. The efficient cleaning was of paramount importance to perform Al speciation at extremely low concentration levels. The repeatability of measurement tested for six consecutive separations of unspiked serum was +/-8.6%. The limits of detection and quantification (based on 3 and 10 s of the blank) were 0.15 and 0.49 ng mL(-1) Al bound to transferrin, respectively. This is the first report on quantitative and reliable speciation of Al in human serum at normal concentration levels.

Development and characterization of methacrylate-based hydrazide monoliths for oriented immobilization of antibodies

Convective interaction media (CIM; BIA Separations) monoliths are attractive stationary phases for use in affinity chromatography because they enable fast affinity binding, which is a consequence of convectively enhanced mass transport. This work focuses on the development of novel CIM hydrazide (HZ) monoliths for the oriented immobilization of antibodies. Adipic acid dihydrazide (AADH) was covalently bound to CIM epoxy monoliths to gain hydrazide groups on the monolith surface. Two different antibodies were afterwards immobilized to hydrazide functionalized monolithic columns and prepared columns were tested for their selectivity. One column was further tested for the dynamic binding capacity.

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 μL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.

Isolation of cell-free DNA from plasma by chromatography on short monolithic columns and quantification of non-apoptotic fragments by real-time polymerase chain reaction

Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.

Trypsin immobilization on an ethylenediamine-based monolithic minidisk for rapid on-line peptide mass fingerprinting studies

The aim of this work was to develop a trypsin-based micro-immobilized enzyme reactor prepared on a monolithic ethylenediamine BIA Separations CIM (convective interaction media) minidisk. The micro-immobilized enzyme reactor (IMER) was integrated in a liquid chromatography system hyphenated to electrospray ionization tandem mass spectrometry to carry out on-line protein digestion and identification. The performance of this IMER was compared with that obtained using a previously developed bioreactor prepared on a conventional CIM ethylenediamine disk and with that of the commercially available Poroszyme immobilized trypsin cartridge. In this work, we showed how different proteins were identified with good recoveries using a digestion time of 10 min only.

Immobilized metal affinity chromatography of histidine-tagged lentiviral vectors using monolithic adsorbents

Histidine-tagged lentiviral vectors were separated from crude cell culture supernatant using labscale monolithic adsorbents by immobilized metal affinity chromatography. The capture capacity, concentration factor, purification factor, and elution efficiency of a supermacroporous cryogel monolith were evaluated against the BIA Separations convective interaction media (CIM) disc, which is a commercial macroporous monolith. The morphology of the polymeric cryogel material was characterised by scanning electron microscopy. Iminodiacetic acid was used as the metal chelating ligand in both monoliths and the chelating capacity for metal ions was found to be comparable. The CIM-IDA-Ni 2+ adsorbent had the greatest capture capacity (6.7 × 10 8 IU/ml of adsorbent), concentration factor (1.3-fold), and elution efficiency (69%). Advantages of the cryogel monoliths included rapid, low pressure processing as well low levels of protein and DNA in the final purified vector preparations.

Trypsin immobilization on three monolithic disks for on-line protein digestion

The preparation and characterization of three trypsin-based monolithic immobilized enzyme reactors (IMERs) developed to perform rapid on-line protein digestion and peptide mass fingerprinting (PMF) are described. Trypsin (EC 3.4.21.4) was covalently immobilized on epoxy, carbonyldiimidazole (CDI) and ethylenediamine (EDA) Convective Interaction Media ® (CIM) monolithic disks. The amount of immobilized enzyme, determined by spectrophotometric measurements at 280 nm, was comprised between 0.9 and 1.5 mg per disk. Apparent kinetic parameters K m * and V max * , as well as apparent immobilized trypsin BAEE-units, were estimated in flow-through conditions using N-α-benzoyl- l-arginine ethyl ester (BAEE) as a low molecular mass substrate. The on-line digestion of five proteins (cytochrome c, myoglobin, α 1-acid glycoprotein, ovalbumin and albumin) was evaluated by inserting the IMERs into a liquid chromatography system coupled to an electrospray ionization ion-trap mass spectrometer (LC-ESI–MS/MS) through a switching valve. Results were compared to the in-solution digestion in terms of obtained scores, number of matched queries and sequence coverages. The most efficient IMER was obtained by immobilizing trypsin on a CIM ® EDA disk previously derivatized with glutaraldehyde, as a spacer moiety. The proteins were recognized by the database with satisfactory sequence coverage using a digestion time of only 5 min. The repeatability of the digestion (R.S.D. of 5.4% on consecutive injections of myoglobin 12 μM) and the long-term stability of this IMER were satisfactory since no loss of activity was observed after 250 injections.

Use of Convective Interaction Media for Analysis of Long‐Nosed Viper Venom

It was confirmed in this study that Vipera ammodytes ammodytes venom samples with a higher content of ammodytoxins (Atxs), basic neurotoxic phospholipases A2, are more lethal, exposing Atxs as one of the major toxic components in the long‐nosed viper venom. In addition, we recently correlated the ability of the venom to produce highly protective antiserum in rabbits with the amount of Atxs in the venom. Here, we developed a rapid, robust, and highly reproducible chromatographic method, based on HPLC separation on Convective Interaction Media to assess the quantity of Atxs in a particular venom sample in order to predict its potential to induce highly protective antiserum in immunized animals.

Anion-exchange chromatography using CIM® DEAE disks as a method of choice for DNA isolation from lecithin

The most important prerequisite for the application of PCR-based methods, among them the detection and quantifi cation of genetically modifi ed organisms (GMOs) is the ability to extract significant amounts of DNA of adequate quality from the sample under investigation. The sample of interest in our work was soybean lecithin with expected low DNA content. The aim of this study was to set up a fast and effective HPLC (High Performance Liquid Chromatography) method using CIM® (Convective Interaction Media, BIA Separations d.o.o., Ljubljana, Slovenia) DEAE (DiEthylAminoEthyl) anion-exchange disk monolithic columns (disks) for the isolation of DNA from soybean lecithin samples. As the reference isolation procedure we used CTAB (CethylTrimethylAmmonium Bromide) method, which is widely used in GMO detection. It was demonstrated, that CIM® DEAE disks allow effi cient isolation of DNA from soybean lecithin. Furthermore, in comparison with the CTAB method, the method was less time-consuming and reduced the use of some aggressive chemicals. The quality of isolated DNA was tested with spectrophotometric analysis, agarose gel electrophoresis and by amplification of soybean specifi c lectin gene with qualitative and real-time PCR. The isolated soybean DNA was of adequate quantity and quality for PCR analysis, even though mostly degraded, present in small amounts and contaminated with some impurities, among them potential PCR inhibitors. The study expanded the applicability of monolithic columns in the isolation of biomolecules from highly processed food materials and their potential use for nucleic acids detection.

Speciation of Al in human serum by convective-interaction media fast-monolithic chromatography with inductively coupled plasma mass spectrometric detection

A new analytical procedure using anion-exchange separation support based on convective-interaction media (CIM) was developed for the speciation of Al in human serum. The separation of proteins was performed on a weak anion-exchange CIM diethylamine (DEAE) fast-monolithic disk. To prevent co-elution of low molecular mass (LMM) Al species with high molecular mass (HMM) Al compounds on CIM disk serum proteins were first separated from LMM-Al species by the use of size exclusion chromatography (SEC). For this purpose 1 mL of serum was injected onto SEC (Superdex 75 HR 10/30) column. Isocratic elution using 0.05 M TRIS–HCl + 0.03 M NaHCO 3 was applied and separation of proteins was followed by UV detection at 278 nm. It was experimentally proven that proteins were eluted in 5.5 mL peak that was collected into a polyethylene cup. A 0.1 mL of the sample aliquot was then injected onto the CIM DEAE disk. The separation of serum proteins was obtained in 10 min by applying linear gradient elution from 100% buffer A (0.05 M TRIS–HCl + 0.03 M NaHCO 3) to 100% buffer B (A + 1 M NH 4Cl) and followed by UV detection at 278 nm. Separated Al species were detected on-line by inductively coupled plasma mass spectrometry (ICP-MS). Well-resolved protein peaks were obtained. It was experimentally proven that 90 ± 3% of Al in spiked serum of renal patient was eluted under the transferrin peak. The proposed speciation procedure removes LMM-Al species and enables reliable determination of the concentration and composition of Al bound to proteins by CIM DEAE-ICP-MS when the concentration of Al in serum is higher than 5 ng mL −1. In comparison to chromatographic columns CIM disks enable faster separation and simpler manipulation during cleaning procedure and coupling to ICP-MS.

Proteomic identification of technologically modified proteins in malt by combination of protein fractionation using convective interaction media and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for the fast separation of proteins and identification of their modifications based on the use of monolithic chromatographic media and mass spectrometric techniques is described. This method has been developed and applied to the analysis of malt proteins and its posttranslational modifications (glycation). Glycation, one of the most common forms of posttranslational modifications (PTM), can be detected in both biological and industrial samples. Our attention was focused on the investigations of possible chemical modifications of water-soluble barley proteins during malting process by combination of anion-exchange chromatography with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The malt extract was directly fractioned by anion-exchange chromatography using short monolithic columns and a linear gradient from 0 to 700 mM NaCl. Sufficient fractionation was obtained for malt sample, which demonstrates the potential of anion-exchange chromatography on this type of column. Proteins in separated fractions were identified by MALDI-TOF/TOF MS. Our proteomic analysis provided the identification of the major proteins present in the malt that were found to be heterogeneously glycated after malting. One of these proteins: nonspecific lipid transfer protein 1 (LTP1) can be used as a marker for characterization of glycation during malting. This protein and its modifications can be easily determined by the developed method.

Development and integration of an immunoaffinity monolithic disk for the on-line solid-phase extraction and HPLC determination with fluorescence detection of aflatoxin B1 in aqueous solutions

The development and characterization of an anti-aflatoxin B1 (anti-AFB1) immunoaffinity monolithic disk is reported. Polyclonal anti-AFB1 was covalently immobilized in batch on an epoxy-activated monolithic Convective Interaction Media (CIM) disk (12 mm × 3 mm i.d.) by a one-step reaction via epoxy groups of the polymer surface. 0.96 mg of antibody were immobilized and the binding capacity of the CIM disk was determined by frontal analysis. The CIM disk was coupled through a switching valve to a reversed-phase column, namely Chromolith Performance RP-18e. A fully automated HPLC method with fluorescence detection for the determination of aflatoxin B1 in aqueous solution was developed. The total analysis time with the integrated system is 46 min and the retention time of AFB1 is approximately 29 min. The binding capacity of the immunoaffinity disk was evaluated in terms of linearity, precision and accuracy of the extraction procedure. The immunoaffinity support was stable after repeated runs.

Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug–protein interactions

In this study, sequential injection affinity chromatography was used for drug–protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy Δ G, enthalpy Δ H) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 × 10 6 M −1 at pH 7.4 and 39 °C for a single binding site. The associated change in enthalpy (Δ H) was −27.36 kcal mol −1 and the change in entropy (Δ S) was −73 cal mol −1 K −1 for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence ( λ ext = 230 nm, λ em = 350 nm) and spectrophotometric detection ( λ = 280 nm).

Fast and efficient separation of immunoglobulin M from immunoglobulin G using short monolithic columns

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20 mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.

Characterization of reversible and pseudo-irreversible acetylcholinesterase inhibitors by means of an immobilized enzyme reactor

The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized on a monolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For this application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective Interaction Media (CIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, low nonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms of action of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver–Burk plot analysis. Analyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak area. AChE-CIM-IMER kinetic parameters ( K m app and v max app ) were derived as well as inhibitory constants ( K i app ) of selected compounds. Moreover, noteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbamoylation and decarbamoylation steps in pseudo-irreversible binding of carbamate derivatives (physostigmine and rivastigmine). AChE-CIM-IMER appeared to be a valid tool to determine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained with the classical methods with the free enzyme. Furthermore, after inactivation by carbamates, activity could be fully recovered and the AChE-CIM-IMER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the first phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracy and reproducibility and with saving of time and materials.