Publications
2003
I. Mihelič, T. Koloini, A. Podgornik
Journal of Applied Polymer Science, Vol. 87, 2326-2334 (2003)
Monolithic stationary phases are becoming increasingly important in the field of liquid chromatography. Methacrylate-based monoliths are produced via free-radical bulk polymerization. The preparation of large-volume monoliths is a major problem because the intensive heat released during polymerization causes distortion of the porous monolithic structure. This work presents experimental measurements of temperature distributions during polymerization in moulds of different sizes and at various experimental conditions. A mathematical model for the prediction of temporal and spatial temperature distribution during the polymerization of methacrylate-based monolithic columns is introduced. The polymerization is described by an unsteady-state heat conduction equation with the generation of heat related to the general kinetics of polymerization. Predictions from the mathematical model are in good agreement with the experimental measurements at different experimental conditions. A method for construction of large-volume monolithic columns is presented and an attempt is made to adopt the developed mathematical model in annular geometry.
P. Milavec Žmak, H. Podgornik, J. Jančar, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1006 (2003) 195–205
Convective Interaction Media (CIM) columns are monolithic columns optimized for the separation of macromolecules. Some of them operate in the axial mode while others operate in the radial mode depending on the column size. In this work we tested the approach suggested by Yamamoto [Biotechnol. Bioeng., 48 (1995) 444] for transfer of gradient methods between columns of different size. A simplified equation for transfer was derived together with a criterion for its application. Separation was evaluated for a standard protein mixture and peroxidase enzymes present in fermentation broth. Salt and pH gradients were applied. Similar resolutions were obtained for each sample on all columns which demonstrates that the proposed approach can be successfully used for method scale-up on this type of column.
M. Merhar, A. Podgornik, M. Barut, M. Žigon, A. Štrancar
J. Sep. Sci. 2003, 26, 322–330
Methacrylate-based monoliths are formed during radical copolymerization as a consequence of the precipitation of polymeric chains from the reaction mixture, which consists of monomers, initiator, and the porogenic solvents. The effect of various methacrylate monomers on the porous structure of the monolith was investigated. Although the chemical structure of the monomers significantly affects the size of the pores and the porosity, the mechanism of pore formation in the case of the precipitation during polymerization is preserved. The porous structure was further correlated with the specific surface area, pressure drop, and dynamic binding capacity of the monoliths studied.
A. Podgornik, M. Barut, A. Štrancar
Encyclopedia of Chromatography DOI: 10.1081/E-ECHR 120016288, 2003
Chromatographic columns are typically several centimeters in length, resulting in a high number of column plates, and, consequently, such columns have high efficiency. These properties allow even very similar molecules to be separated. This is especially true for smaller molecules, where the separation is based on selective migration. For large molecules, a different separation mechanism is usually required. Large molecules normally interact with the matrix at several binding sites. Consequently, their adsorption isotherms are very steep, almost rectangular. For such molecules, there exists only a very narrow mobile phase range within which they interact with the active moieties on the stationary phase, but are not irreversibly retained. To elute them from the matrix, a change of the mobile phase composition is required. Therefore the separation is based upon the selective elution and requires the use of gradient chromatographic methods. For this type of separation, the column length is less important and the efficient separations can be achieved even with extremely short columns.
P. Kramberger, D. Glover, A. Štrancar
American Biotechnology Laboratory, 2003, 27-28
Research in molecular and cell biology has shown that macromolecules such as pDNA and virus vectors, together called nanoparticles, have the potential to assist in the prevention and treatment of some human diseases. The most important step in their production is the downstream processing (isolation and cleaning). Precipitation, ultrafiltration, and LC techniques are the most widely used for these purposes, but only LC can purify the product so that it is recognized as safe for therapeutic use.
Apart from reduced yield, downstream processing can cause minor or even major modifications in the structure of the biomolecule. Usually these modifications do not affect the activity of the product, but may change its antigenicity. Minimizing these changes to maintain product safety is the main objective in the downstream processing of nanoparticles. For the efficient isolation of labile biomolecules, liquid chromatographic supports should provide fast and efficient separation in order to decrease biomolecule degradation; have high, preferably flow-unaffected capacity and resolution; and exhibit low backpressure. They should be stable, even if harsh conditions are applied during sanitation (e.g., 1 MNaOH), and should be easy to handle and operate.
CIM® (Convection Interaction Media) monolithic chromatographic columns (BIA Separations, Ljubljana, Slovenia) meet all of these requirements. This application note will discuss the columns and their use on human models and plant viruses and pDNA.
I. Mihelič, A. Podgornik, T. Koloini
Journal of Chromatography A, 987 (2003) 159–168
This work investigates the influence of temperature on the binding capacity of bovine serum albumin (BSA), soybean trypsin inhibitor and l-glutamic acid to a CIM® (DEAE) weak anion-exchange disk monolithic column. The binding capacity was determined experimentally under dynamic conditions using frontal analysis. The effect on the dynamic binding capacity of dimers present in the BSA solution has been evaluated and a closed-loop frontal analysis was used to determine the equilibrium binding capacities. The binding capacity for both BSA and soybean trypsin inhibitor increased with increasing temperature. In the case of l-glutamic acid, an increase in the binding capacity was observed with temperature up to 20 °C. A further increase in temperature caused a decrease of the dynamic binding capacity.
M. Vodopivec, A. Podgornik, M. Berovič, A. Štrancar
Journal of Chromatography B, 795 (2003) 105-113
The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis–Menten constant Km and the turnover number k3 of the immobilized enzymes were found to be flow-unaffected. Furthermore, the Km values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.
2002
A. Podgornik, M. Barut, S. Jakša, J. Jančar, A. Štrancar
Journal of Liquid Chromatography & Related Technologies Vol. 25, No. 20, pp. 3097–3114, 2002
Convective Interaction Media® (CIM) disk monolithic columns are specific among the chromatographic columns because of their monolithic structure and extremely short column length. In this work, HETP values and Z factors for different groups of molecules—proteins, DNA, oligonucleotides, peptides, and organic acids on strong anion exchange CIM disk monolithic columns were determined. Results are discussed in terms of the molecule structures and applied to develop different approaches for successful separation of abovementioned group of molecules on these types of columns.
R. Hahn, M. Panzer, E. Hansen, J. Mollerup, A. Jungbauer
Separation Science and Technology, 37(7), 1545–1565 (2002)
The mass transfer properties of polyglycidylmethacrylate–ethylenedimethacrylate monolithic ion-exchangers (convective interaction media disks) were evaluated. As a reference material, the particulate ion-exchanger Source 30 was selected. The model proteins lysozyme, bovine serum albumin, and IgG were loaded at different concentrations and velocities. The mass transfer zones obtained with the monoliths were affected by neither the linear flow velocity nor the protein concentration in the mobile phase. The reduced height equivalent to one theoretical plate (HETP) of monoliths were independent of the reduced velocity. This was not the case for the particulate material.
A. Štrancar, A. Podgornik, M. Barut, D. Glover
BIOforum International 3/2002
In adsorptive chromatographic modes, the slope of the capacity factor k' (defined as the molar ratio of the separated compound in the stationary phase and the mobile phase) plot versus composition of the mobile phase is very steep. Up to a certain composition of the mobile phase, k' is so high that the protein is bound to the stationary phase and does not move along the column. Reaching a defined point, a small change of the mobile phase composition causes a rapid decrease in k' to a value near zero. At this point, the protein dissolves in the mobile phase and passes through the column practically without any retention. In other words, the protein remains adsorbed at the top of the column until the eluting power of the mobile phase reaches the point at which a small change in the composition of the mobile phase causes the movement of the protein without any retention. One can also speak about selective elution of the compound. As a result of this process, even very short columns can provide very good separations and recovery, while longer columns might cause problems due to unspecific binding, product degradation and minor changes in the structure of the protein which increase with the length of the column. On the other hand, short-beds are very difficult to pack with particles and form channels which eliminate the resolution power of the column. Monolithic supports offer an ideal solution to avoid most of these problems.
K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer
J. Comb. Chem. 2002, 4, 33-37
Solid-phase peptide synthesis was performed on glycidyle methacrylate-co-ethylene dimethacrylate monoliths using Fmoc chemistry. The native epoxy groups were amino-functionalized by reaction with ethylenediamine or ammonia ions. A peptide directed against human blood coagulation factor VIII was synthesized as a model peptide. Amino acid analysis revealed the correct amino acid ratio as present in the sequence. The ligand density of 5 μmol/mL was equal to that achieved with conventional peptide immobilization via epoxy groups. These supports were directly used as peptide affinity chromatography matrixes. The functionality of the CIM monolithic supports was proven by affinity chromatography of factor VIII. The ammonia-functionalized support performed with low hydrophobicity and did not show unspecific adsorption of proteins.
K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer
Biotechnology and Bioengineering 79 (2002) 733-740
Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM® disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 733–740, 2002.
T. Tennikova, A. Štrancar
LabPlus international February - March 2002, Volume 16
Monolithic supports are a novel generation of stationary phases that can be used for liquid and gas chromatography, capillary electrochromatography, bioconversions, as well as supports for solid phase synthesis. In contrast to individual particles packed into chromatographic columns, monolithic supports are cast as continuous homogeneous phases. They provide high rates of mass transfer at lower pressure drops and enable much faster separations. In addition to the speed, the nature of the pores allows easy permeability for large molecules. Monolithic supports are thus the method of choice for the separation of proteins, oligonucleotides, and nanoparticles such as pDNA and viruses. In this article we review the application of the monlithic columns to bioaffinity chromatography.
H. Podgornik, A. Podgornik
Enzyme and Microbial Technology 31 (2002) 855–861
The possibility of covalent attachment of LiP H2 and LiP H8 to CIM monoliths was studied. Due to negligible diffusional resistance, they can be useful tools to study characteristics of the immobilized lignin peroxidase (LiP). Immobilization to epoxy groups was performed using alkaline conditions (borate-phosphate buffer; pH 7.5). Characteristics of immobilized LiP were compared and factors that influence their biologic activity were evaluated using flow through experiments. Enzyme kinetics was determined via oxidation of veratryl alcohol (VA) into veratraldehyde (Vald). While VA oxidation rate increased by increasing flow rate (up to 1.5 ml/min) for LiP H2, it was almost constant in a wide flow rate range for LiP H8. This observation together with the stepwise deactivation of the enzyme by consecutive experiments was ascribed to accumulation of the formed Vald inside the support. Calculated kinetic parameters showed 3–5 times higher Km value for VA for both tested isoforms in comparison to free enzyme. The catalytic constant was found to be approximately 0.5 s-1 for both isoforms. Immobilized LiP H8 was used for decolorization of azo dye Mahogany.
K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer
Biotechnology and Bioengineering 79 (2002) 733-740
Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM® disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions.
A. Podgornik, T. B. Tennikova
Advances in Biochemical Engineering/ Biotechnology, 2002, Vol. 76, 167-206
In the last decade there were many papers published on the study of enzyme catalyzed reactions performed in so-called chromatographic reactors. The attractive feature of such systems is that during the course of the reaction the compounds are already separated, which can drive the reaction beyond the thermodynamic equilibrium as well as remove putative inhibitors. In this chapter, an overview of such chromatographic bioreactor systems is given. Besides, some immobilization techniques to improve enzyme activity are discussed together with modern chromatographic supports with improved hydrodynamic characteristics to be used in this context.
H. Podgornik, A. Podgornik
Enzyme and Microbial Technology 31 (2002) 855–861
The possibility of covalent attachment of LiP H2 and LiP H8 to CIM monoliths was studied. Due to negligible diffusional resistance, they can be useful tools to study characteristics of the immobilized lignin peroxidase (LiP). Immobilization to epoxy groups was performed using alkaline conditions (borate-phosphate buffer; pH 7.5). Characteristics of immobilized LiP were compared and factors that influence their biologic activity were evaluated using flow through experiments. Enzyme kinetics was determined via oxidation of veratryl alcohol (VA) into veratraldehyde (Vald). While VA oxidation rate increased by increasing flow rate (up to 1.5 ml/min) for LiP H2, it was almost constant in a wide flow rate range for LiP H8. This observation together with the stepwise deactivation of the enzyme by consecutive experiments was ascribed to accumulation of the formed Vald inside the support. Calculated kinetic parameters showed 3–5 times higher Km value for VA for both tested isoforms in comparison to free enzyme. The catalytic constant was found to be approximately 0.5 s−1 for both isoforms. Immobilized LiP H8 was used for decolorization of azo dye Mahogany.
2001
I. Mihelič, M. Krajnc, T. Koloini, A. Podgornik
Ind. Eng. Chem. Res. 2001, 40, 3495-3501
Monolithic stationary phases are becoming more and more important in the field of liquid chromatography, because they enable extremely fast separations. Methacrylate-based monoliths are produced via a free-radical bulk polymerization of glycidyl methacrylate and ethylene dimethacrylate using a benzoyl peroxide as an initiator. Preparation of large monoliths represents a big problem because of the heat release during the polymerization, which consequently leads to the distortion of the structure. A closer investigation of the polymerization, using differential scanning calorimetry, was performed in order to determine global kinetic parameters. A multiple heating rate method, based on the work of Ozawa, Flynn, and Wall, was applied for estimation of the values of the apparent activation energy, preexponential factor, and reaction order. Global polymerization kinetics is of first order with A = 1.681 × 109 s-1 and Ea,app = 81.5 kJ/mol, where the heat of polymerization is approximately 190 J/g. In addition, the influence of air and nitrogen atmosphere on polymerization is presented.
M. Merhar, A. Podgornik, M. Barut, S. Jakša, M. Zigon, A. Štrancar
J. Liq. Chrom. & Rel. Technol., 24(16), 2429-2443 (2001)
Monoliths have already proven to be efficient chromatographic supports for the separation of various types of molecules. In this paper, the characterization of the novel reversed-phase support, CIM® RP-SDVB disk monolithic column is presented.
Using a 3 mm long RP-SDVB disk monolithic column, excellent separation of proteins within a very short time was achieved. The pressure drop observed on the column was considerably low (few bars), even at flow rates of the mobile phase up to 30 mL/min. Due to the low pressure drop, the use of high flow rates was preferred since they did not influence the quality of the gradient separation. The separation of test proteins was performed within only 14 seconds; faster separations were limited by the configuration of the HPLC system.
Furthermore, RP-SDVB disk monolithic columns were applied for fast separation of peptides. Five peptides of different lengths and composition were successfully separated in a very short time.
Finally, the preparative purification on the laboratory scale of the complex sample of oligodeoxynucleotide within a range of 1 minute demonstrates practical applicability of these columns.
D. Josić, A. Buchacher
J. Biochem. Biophys. Methods 49 (2001) 153–174
Monoliths are useful chromatographic supports, as their structure allows improved mass transport. This results in fast separation. Once the ligand of interest has been immobilized, chromatographic separation can also be accomplished in affinity mode. Ligands with low molecular mass have been shown to be the easiest to immobilize. Nowadays, ligands with low molecular mass are often designed by combinatorial chemical techniques. In addition, many applications have been described where ligands with high molecular mass, such as Proteins A and G, antibodies, lectins and receptors are used.
The immobilization of an enzyme on the monolithic support creates a flow-through reactor. Small proteins, such as carbonic anhydrase, can be directly immobilized on the support. However, in the case of large molecules, the active center of the enzyme is no longer accessible at all or only to a limited degree. An improvement can be achieved by introducing a spacer, which allows maximum enzymatic conversion. Fast conversion of substrates with high molecular mass has been investigated with immobilized trypsin. It was shown that in case of high-molecular-mass substrates, the conversion rate depends very much on the flow-rate. Most applications described have been performed on an analytical or semi-preparative scale. However, the technical problems of up-scaling are close to being definitely solved, enabling enzymatic conversion on a preparative scale in the future.