Publications
2008
E. S. Sinitsyna, E. N. Vlasova, E. G. Vlakh, T. B. Tennikova
Russian Journal of Applied Chemistry, 2008, Vol. 81, No. 8, pp. 1403–1409
Copolymers containing aldehyde, succinimidyl carbonate, and imidazolecarbamate groups were prepared by polymer-analogous transformations of epoxy groups of a macroporous monolithic polymeric support derived from glycidyl methacrylate and ethylene glycol dimethacyrlate. The effect of certain parameters on the course of the copolymer modification and immobilization of a protein on the surface of the polymeric support was studied. The possibility of using the matrices obtained for development of biorecognizing systems was examined.
M. Barut, A. Podgornik, L. Urbas, B. Gabor, P. Brne, J. Vidič, S. Plevčak, A. Štrancar
J. Sep. Sci. 2008, 31, 1867 – 1880
This review describes the novel chromatography stationary phase – a porous monolithic methacrylate-based polymer – in terms of the design of the columns and some of the features that make these columns attractive for the purification of large biomolecules. We first start with a brief summary of the characteristics of these large molecules (more precisely large proteins like immunoglobulins G and M, plasmid deoxyribonucleic acid (DNA), and viral particles), and a list of some of the problems that were encountered during the development of efficient purification processes. We then briefly describe the structure of the methacrylate-based monolith and emphasize the features which make them more than suitable for dealing with large entities. The highly efficient structure on a small scale can be transferred to a large scale without the need of making column modifications, and the various approaches of how this is accomplished are briefly presented in this paper. This is followed by presenting some of the examples from the bioprocess development schemes, where the implementation of the methacrylate-based monolithic columns has resulted in a very efficient and productive process. Following this, we move back to the analytical scale and demonstrate the efficiency of the monolithic column – where the mass transfer between the stationary and mobile phase is greatly enhanced – for the in-process and final control of the new therapeutics. The combination of an efficient structure and the appropriate hardware results in separations of proteins with residence time less than 0.1 s.
F. Smrekar, M. Ciringer, M. Peterka, A. Podgornik, A. Štrancar
Journal of Chromatography B, (2007)
Phages are gaining importance due to their wide usage. In this work strong anion exchange monolithic chromatographic column was used for single step phage purification. Most of the proteins and DNA were removed and recovery of approximately 70% of infective virus was reproducibly achieved. 30 ml of phage sample was purified in around 10 min.
I. V. Kalashnikova, N. D. Ivanova, T. B. Tennikova
Russian Journal of Applied Chemistry, 2008, Vol. 81, No. 5, pp. 867-873
A simple virus-cell complementary model system can be obtained using polymer-analogous reactions of the epoxy groups of glycidyl methacrylate-ethylene glycol dimethacrylate monolithic macroporous polymeric support and of the carboxy groups of styrene-methyl methacrylate polymeric nanospheres. The effect of thus designed microenvironment on the affinity binding parameters of virus-mimicking nanoparticles with the functionalized sorbent surface is studied by high-performance monolithic disk affinity chromatography.
S. Likić, G. Rusak, M. Krajačić
Journal of Chromatography A, 1189 (2008) 451–455
High-performance liquid chromatography was developed for further separation of double-stranded (ds) RNAs obtained by CF-11 cellulose chromatography from plants infected with satellite associated cucumber mosaic virus. Fractions separated by monolithic polymer column, especially applicable for nucleic acid analyses, were identified electrophoretically and confirmed with a polymerase chain reaction test. Once standardized, the method has revealed clear evidence of satellite presence without precipitation and electrophoresis. According to demonstrated sensitivity, its application in the preliminary diagnostics of field samples is also predictable. Principally, it can be used as a powerful preparative approach resulting in highly pure satellite dsRNA for further analyses.
C. Delattre, P. Michaud, M. A. Vijayalakshmi
Journal of Chromatography B, 861 (2008) 203–208
Fast production and purification of α-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 °C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.
2007
R. Hahn, A. Tscheliessnig, P. Bauerhansl, A. Jungbauer
J. Biochem. Biophys. Methods 70 (2007) 87–94
Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.
R. Skudas, B. A. Grimes, E. Machtejevas, V. Kudirkaite, O. Kornysova, T. P. Hennessy, D. Lubda, K. K. Unger
Journal of Chromatography A, 1144 (2007) 72-84(2007) 72 - 84
In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of ∼25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18 < C8 < C4. The resolution of proteins and peptides by reversed-phase gradient liquid chromatography on n-octadecyl, n-octyl, and n-butyl bonded monolithic silica columns is optimized. The results obtained imply the use of acetonitrile concentration gradient up to 75% for n-octadecyl and n-octyl bonded monolithic silica columns, and the use of acetonitrile concentration gradient up to 85% for n-butyl bonded monolithic silica columns. With the respect to the gradient times and flow rates, the optimum conditions are the best with n-octyl and n-butyl bonded monolithic silica columns, where the range of optimum gradient times is up to ∼30 min and mobile phase flow rates in the range of 0.5–1 ml/min. Consequently, the best performance towards peak resolution is obtained with n-octyl bonded monolithic silica column with the respect to low concentration of organic phase gradient, fast separations and low solvent consumptions due to low flow rates.
J. Vidič, A. Podgornik, J. Jančar, V. Frankovič, B. Košir, N. Lendero, K. Čuček, M. Krajnc, A. Štrancar
Journal of Chromatography A, 1144 (2007) 63–71(2007) 63 – 71
Chemical and chromatographic stability of methacrylate-based monolithic columns bearing 3-N,N-diethylamino-2-hydroxypropyl (DEAE) and quarternary amine (QA) groups was studied. The leakage products from both monolithic columns were determined and the leakage of amines has been quantified in alkali solutions. Monolithic columns bearing QA functional groups being exposed to 1 M sodium hydroxide solution for up to 3 months caused reduction of ion-exchange groups for approximately 12%, while for DEAE monolithic columns was only around 3% in 1 year. In 0.1 M NaOH and 20% ethanol degradation was significantly lower. The main leaking compound from DEAE monolith was found to be 3-(diethylamino)-1,2-propanediol and 2,3-dihydroxypropyltrimethylammonium salt for QA monolith. During repeated 50 cleaning-in-place (CIP) cycles, no changes in chromatographic properties were detected.
B. A. Grimes, R. Skudas, K. K. Unger, D. Lubda
Journal of Chromatography A, 1144 (2007) 14-29(2007) 14-29
In this work, a parallel pore model (PPM) and a pore network model (PNM) are developed to provide a state-of-art method for the calculation of several characteristic pore structural parameters from inverse size-exclusion chromatography (ISEC) experiments. The proposed PPM and PNM could be applicable to both monoliths and columns packed with porous particles. The PPM and PNM proposed in this work are able to predict the existence of the second inflection point in the experimental exclusion curve that has been observed for monolithic materials by accounting for volume partitioning of the polymer standards in the macropores of the column. The appearance and prominence of the second inflection point in the exclusion curve is determined to depend strongly on the void fraction of the macropores (flow-through pores), (b) the nominal diameter of the macropores, and (c) the radius of gyration of the largest polymer standard employed in the determination of the experimental ISEC exclusion curve. The conditions that dictate the appearance and prominence of the second inflection point in the exclusion curve are presented. The proposed models are applied to experimentally measured ISEC exclusion curves of six silica monoliths having different macropore and mesopore diameters. The PPM and PNM proposed in this work are able to determine the void fractions of the macropores and silica skeleton, the pore connectivity of the mesopores, as well as the pore number distribution (PND) and pore volume distribution (PVD) of the mesopores. The results indicate that the mesoporous structure of all materials studied is well connected as evidenced by the similarities between the PVDs calculated with the PPM and the PNM, and by the high pore connectivity values obtained from the PNM. Due to the fact that the proposed models can predict the existence of the second inflection point in the exclusion curves, the proposed models could be more applicable than other models for ISEC characterization of chromatographic columns with small diameter macropores (interstitial pores) and/or large macropore (interstitial pore) void fractions. It should be noted that the PNM can always be applied without the use of the PPM, since the PPM is an idealization that considers an infinitely connected porous medium and for materials having a low (<6) pore connectivity the PPM would force the PVD to a lower average diameter and larger distribution width as opposed to properly accounting for the network effects present in the real porous medium.
I. Junkar, T. Koloini, P. Krajnc, D. Nemec, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1144 (2007) 48-54(2007) 48-54
Today, monoliths are well-accepted chromatographic stationary phases due to several advantageous properties in comparison with conventional chromatographic supports. A number of different types of monoliths have already been described, among them recently a poly(high internal phase emulsion) (PolyHIPE) type of chromatographic monoliths. Due to their particular structure, we investigated the possibility of implementing different mathematical models to predict pressure drop on PolyHIPE monoliths. It was found that the experimental results of pressure drop on PolyHIPE monoliths can best be described by employing the representative unit cell (RUC) model, which was originally derived for the prediction of pressure drop on catalytic foams. Models intended for the description of particulate beds and silica monoliths were not as accurate. The results of this study indicate that the PolyHIPE structure under given experimental condition is, from a hydrodynamic point of view, to some extent similar to foam structures, though any extrapolation of these results may not provide useful predictions of pressure versus flow relations and further experiments are required.
K. Isobe, Y. Kawakami
Journal of Chromatography A, 1144 (2007) 85-89(2007) 85-89
A convection interaction media (trade name CIM, Sartorius BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.
S. Laschober, M. Sulyok, E. Rosenberg
Journal of Chromatography A, 1144 (2007) 55-62(2007) 55-62
The present work aims at the optimisation of the synthesis of methyl-silsesquioxane monolithic capillary columns using a sol–gel based protocol. The influence of reaction conditions such as temperature, reaction mixture composition and catalyst concentration has been examined. The morphology of the products was studied by scanning electron microscopy and nitrogen adsorption. Monolithic capillary columns were obtained with a skeleton-like structure with open pores. Pore diameters vary from 0.8 to 15 μm, diameters of the xerogel network vary from 0.4 to 12 μm, respectively. Specific surface areas up to 334 m2/g have been observed, however, many materials did not possess areas above few m2/g which represents the limit of detection of the nitrogen porosimetry measurements. Excellent adhesion to the capillary wall was observed in all cases, and drying was possible at ambient conditions without the formation of cracks.
I.Vovk, B. Simonovska
Journal of Chromatography B, 849 (2007) 337-343
The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; Mr 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with Mr 82 kDa) and a basic protein with Mr 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM®) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2 M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5 M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with Mr 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH3CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5 mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.
I. Vovk, B. Simonovska
Journal of Chromatography A, 1144 (2007) 90-96(2007) 90-96
An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, Sartorius BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 μg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 μg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5 mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.
E. Machtejevas, S. Andrecht, D. Lubda, K. K. Unger
Journal of Chromatography A, 1144 (2007) 97-101
The following particulate and monolithic silica columns were implemented in a fully automated and flexible multidimensional LC/MS system with integrated sample clean-up, to perform the analysis of endogeneous peptides from filtered urine and plasma samples: restricted access sulphonic acid strong cation-exchanger (RAM-SCX) for sample clean-up, RP 18 Chromolith guard columns as trap columns and 100 μm I.D. monolithic RP 18 fused silica capillary columns as last LC dimension. The results show sufficient overall system reproducibility and repeatability. Implementation of monolithic silica columns added an additional flexibility with respect to flow rate variation and adjustment due to the low column back pressures. Also, monolithic columns showed a lower clogging rate in long-term usage for biological samples as compared to particulate columns. The applied system set-up was tested to be useful for the routine peptide screening in search of disease biomarkers.
P. Kramberger, M. Peterka, J. Boben, M. Ravnikar, A. Štrancar
Journal of Chromatography A, 1144 (2007), pg. 143–149
Drawbacks of conventional virus purification methods have led to the development of new, mostly chromatography-based methods. Short monolithic columns are stationary phases intended for purification of large molecules. In this work efficient chromatographic purification of tomato mosaic virus (ToMV) from plant material is described. Based on short monolithic column, the purification process was shortened from 5 days to 2 hours. High viral purity was achieved and recovery of chromatographic step was up to 90%. In addition, these columns enabled preliminary quantification of the virus in just a few minutes, much faster than other quantification methods (e.g. enzyme-linked immunosorbent assay or real-time polymerase chain reaction) which take 1–2 days. These results demonstrate the potential of short monolith column technology for purification and analysis of different viruses.
J. Boben, P. Kramberger, N. Petrovič, K. Cankar, M. Peterka, A. Štrancar, M. Ravnikar
European Journal of Plant Pathology (2007) 118:59-71
A quantitative RT real-time PCR method was developed for the detection and quantification of Tomato mosaic virus (ToMV) in irrigation waters. These have rarely been monitored for the presence of plant pathogenic viruses, mostly due to the lack of efficient and sensitive detection methods. The newly developed method presented here offers a novel approach in monitoring the health status of environmental waters. ToMV was reliably detected at as low as 12 viral particles per real-time PCR reaction, which corresponds to the initial concentration of approximately 4.2 × 10-10 mg (6,300 viral particles) of ToMV per ml of sample. The sensitivity of the method was further improved by including the Convective Interaction Media® (CIM) monolithic chromatographic columns for quick and efficient concentration of original water samples. Seven out of nine water sources from different locations in Slovenia tested positive for ToMV, after concentrating the sample. Four samples tested ToMV-positive without the concentrating procedure. The presence and integrity of infective ToMV particles in the original sample, as well as in the chromatographic fraction, was confirmed using different methods from test plants, DAS ELISA to electron microscopy and real-time PCR. In this study, we propose a unique and simple diagnostic scheme for rapid, efficient, and sensitive monitoring of irrigation waters that could also be adopted for other plant, human or animal viruses.
M. Peterka, D. Glover, P. Kramberger, M. Banjac, A. Podgornik, M. Barut, A. Štrancar
BioProcessing Journal, March/April 2005
The last 30 years have seen rapid and dramatic developments in recombinant DNA technology and the related biological sciences. In 1972, Paul Berg's group used restriction enzymes to cut DNA in half and then used ligases to stick the pieces of the DNA back together. By doing this, they produced the first recombinant DNA. Within a year, the first genetically engineered bacterium existed. A little more than ten years later, recombinant human insulin was approved for diabetic patients and became the first recombinant healthcare product. Before the end of the 1980s, the first gene therapy trial had occurred. Today, a large number of recombinant proteins are used as marketed drugs and even more are in clinical trials targeting a wide range of diseases.
R. Nicoli, N. Gaud, C. Stella, S. Rudaz, J.-L. Veuthey
Journal of Pharmaceutical and Biomedical Analysis 48 (2008) 398–407
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, carboxy imidazole (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, α1-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.