Publications
2002
N. D. Ostryanina, G. P. Vlasov, T. B. Tennikova
Journal of Chromatography A, 949 (2002) 163–171
High-performance monolithic disk chromatography (HPMDC), including its affinity mode, is a very efficient method for fast separations of biological molecules of different sizes and shapes. In this paper, protein and peptide ligands, immobilized on the inner surface of thin, monolithic supports (Convective Interaction Media or CIM® disks), have been used to develop methods for fast, quantitative affinity fractionation of pools of polyclonal antibodies from blood sera of rabbits, immunized with complex protein–peptide conjugates. The combination of several disks with different affinity functionalities in the same cartridge enables the separation of different antibodies to be achieved within a few minutes. The apparent dissociation constants of affinity complexes were determined by frontal analysis. Variation of elution flow rate over a broad range does not affect the affinity separation characteristics. Indifferent synthetic peptides used as biocompatible spacers do not change the affinity properties of the ligands. The highly reproducible results of immunoaffinity HPMDC are compared with data obtained by widely used enzyme-linked immunosorbent assay.
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.
R. Hahn, A. Podgornik, M. Merhar, E. Schallaun, A. Jungbauer
Anal. Chem. 2001, 73, 5126-5132
An affinity monolith with a novel immobilization strategy was developed leading to a tailored pore structure. Hereby the ligand is conjugated to one of the monomers of the polymerization mixture prior to polymerization. After the polymerization, a monolithic structure was obtained either ready to use for affinity chromatography or ready for coupling of additional ligand to further increase the binding capacity. The model ligand, a peptide directed against lysozyme, was conjugated to glycidyl methacrylate prior to the polymerization. With this conjugate, glycidyl methacrylate, and ethylene dimethacrylate, a monolith was formed and tested with lysozyme. A better ligand presentation was achieved indicated by the higher affinity constant compared to a conventional sorbent.
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.
I. Mihelič, T. Koloini, A. Podgornik, M. Barut, A. Štrancar
Acta Chim. Slov. 2001, 48, 551-564
Monolithic stationary phases are becoming very important field of liquid chromatography. Methacrylate based CIM Convective Interaction Media® monolithic columns and are produced via radical polymerization, which results in a rigid and chemically very stable porous monolithic structure. Some characteristics of small-scale monolithic columns and an example of extremely fast separation of biomolecules are presented in the paper. However, the preparation of large and homogeneous monolithic columns represents a big problem, because the heat released during the polymerization causes distortion of the monolithic structure. A mathematical model employing the polymerization kinetics for the prediction of the temperature profiles and a comparison with the experimental results is presented with the emphasis on the conversion and the rate od the heat release profiles. Finally, the characteristics of a large-scale monolithic column are presented.
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.
H. Podgornik, M. Stegu, A. Podgornik, A. Perdih
FEMS Microbiology Letters 201 (2001) 265-269
High initial Mn(II) concentration results in accumulation of a Mn(III) tartrate complex in the growth medium of Phanerochaete chrysosporium. Since Mn(III) is the major oxidant in ligninolysis by manganese peroxidase, the role of accumulated complex should not be neglected when degradation experiments by a crude culture filtrate are performed. To study the Mn(III) complex oxidative potential it was isolated by absorption to polyamide followed by desorption with an alkaline methanol solution. High performance liquid chromatography analysis and atomic absorption spectroscopy confirmed that the isolate was Mn(III) tartrate. Oxidation of 2,2′-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) was used for testing the temperature and pH stability of the isolate that also intensively oxidized 2,6-dimethoxyphenol. In comparison with the non-isolated complex in the culture filtrate, the isolate showed increased temperature and pH stability. The oxidative potential of the isolated Mn(III) tartrate was additionally tested by decolorization of the synthetic dye Indigo carmine.
2000
R. Hahn, A. Jungbauer
Analytical Chemistry, 7.2. 2000, (4853-4858)
Monoliths are stationary phases cast as a continuous medium which are interlaced by flow channels ramified with micropores. Pulse response experiments with bovine serum albumin as a model protein were applied for testing polymethacrylate-based monoliths, resulting in peak broadening that practically was not influenced by the chromatographic velocity. An empirical model was developed to describe peak broadening, allowing a term to account for the pore convection and a term for the pore diffusion. A diffusional distance lower than 10 nm was estimated. This corresponds to values observed with monodisperse 1-μm particles. Systematic investigations by changing the response time of the detector showed that the full potential of the monoliths could not be exploited, since the currently available chromatography systems are the limiting factor regarding the speed of data acquisition and virtual peak broadening by the infinite length of the detector. Inertia of the liquid and synchronization between liquid handling and electronic control introduced an additional disturbance. At the lowest possible response time, reliable peak data could be obtained up to a velocity of 35 cm/min. The pressure drop along the continuous bed was much smaller compared to a conventionally packed bed. Different flow patterns and significantly reduced eddy vortexes may be responsible for the high specific permeability.
K. Amatschek, R. Necina, R. Hahn, E. Schallaun, H. Schwinn, D. Josić, A. Jungbauer
Journal of Separation science, 23 (2000) 47-58
FVIII is a very complex molecule of great therapeutic significance. It is purified by a sequence of chromatographic steps including immunoaffinity chromatography. A peptide affinity chromatography method has been developed using peptides derived from a combinatorial library. Spot technology using cellulose sheets has been applied for this purpose. The dual positional scanning strategy was used for identification of the amino acids in random positions. Approximately 5000 possible candidates found in the first screening round were reduced to a panel of 36. Six candidates have been selected empirically. Five peptides seem to be directed against the light chain of FVIII, one peptide seems to be directed against the heavy chain. The peptides have been immobilized on conventional beaded material and CIM polymethacrylate monoliths. Much better performance with respect to capacity and selectivity has been observed with the monolithic material. Exposure of the ligand and its ensuing accessibility are responsible for these properties.
L. G. Berruex, R. Freitag, T. B. Tennikova
Journal of Pharmaceutical and Biomedical Analysis 24 (2000) 95–104
A novel biochromatographic principle is introduced taking the quantitative analysis of affinity interactions between antibodies and immobilized group specific ligands (protein A, G, and L) as example. The name high performance monolith affinity chromatography (HPMAC) is proposed for this technique. HPMAC uses rigid, macroporous monoliths, so-called convective interaction media (CIM™)-disks, as stationary phase. An optimized procedure is described for the covalent immobilization of the group specific affinity ligands to such disks. The binding of polyclonal bovine IgG and a recombinant human antibody (type IgG1-κ) to all affinity disks is discussed. An essential feature of HPMAC is its compatibility to unusually high mobile phase flow rates (>4 ml/min). Chromatographic experiments are thus completed within seconds without significant loss in binding capacity and retentive power. This makes HPMAC a promising tool for applications in fast process monitoring or screening. As an example for the former, the direct quantitative isolation of recombinant antibodies from serum-free culture supernatant is demonstrated.
H. LeThanh, B. Lendl
Analytica Chimica Acta 422 (2000) 63–69
A fully automated method for the rapid determination of organic acids (citric-, malic- and tartaric acid) and sugars (glucose, fructose, and sucrose) in soft drinks by sequential injection Fourier transform infrared (FTIR) spectroscopy is presented. A convective interaction media (CIM) disc carrying quaternary amino moieties was added as a solid phase extraction column to the flow system. Upon injection of a sample the organic acids were completely retained on the CIM disc whereas sugars passed to the flow cell. The organic acids were subsequently eluted by injection of an alkaline (pH 8.5) 1 M sodium chloride solution and recorded in their fully deprotonated form as a second flow injection peak. In both cases, the FTIR spectra corresponding to the peak maxima were selected for data evaluation. Two partial least squares models, one for sugars and the other for organic acids, were constructed based on the analysis of standards containing all six analytes. The developed method was applied to natural samples yielding results which were in good agreement with those obtained by an external reference method (enzymatic test kits). Deviations in the results were 3.4. and 4.1% for citric and malic acid and ranged from 4.7–5.1% for the sugars. The developed method is characterized by its short analysis time, experimental simplicity and its potential applications in routine analysis and process control.
L. G. Berruex, R. Freitag, T. B. Tennikova
Journal of Pharmaceutical and Biomedical Analysis 24 (2000) 95–104
A novel biochromatographic principle is introduced taking the quantitative analysis of affinity interactions between antibodies and immobilized group specific ligands (protein A, G, and L) as example. The name high performance monolith affinity chromatography (HPMAC) is proposed for this technique. HPMAC uses rigid, macroporous monoliths, so-called convective interaction media (CIM™)-disks, as stationary phase. An optimized procedure is described for the covalent immobilization of the group specific affinity ligands to such disks. The binding of polyclonal bovine IgG and a recombinant human antibody (type IgG1-κ) to all affinity disks is discussed. An essential feature of HPMAC is its compatibility to unusually high mobile phase flow rates (>4 ml/min). Chromatographic experiments are thus completed within seconds without significant loss in binding capacity and retentive power. This makes HPMAC a promising tool for applications in fast process monitoring or screening. As an example for the former, the direct quantitative isolation of recombinant antibodies from serum-free culture supernatant is demonstrated.
M. Vodopivec, M. Berovič, J. Jančar, A. Podgornik, A. Štrancar
Analytica Chimica Acta 407 (2000) 105-110
A new polymeric macroporous material, Convective Interaction Media (CIM) was applied as a support for glucose oxidase (GOD) immobilisation. CIM epoxy disks with the immobilised enzyme were integrated as an enzyme reactor in a flow injection analysis (FIA) system and applied to on-line monitoring of glucose during cultivation of Saccharomyces cerevisiae and citric acid production by Aspergillus niger. The developed CIM GOD disk–FIA system exhibited good signal reproducibility and satisfactory long-term stability with a linear response in the range 10–200 mg l-1. The CIM disk applied as an enzyme reactor proved to be a successful replacement for conventionally used packed-bed columns and as such it was well suited for on-line monitoring of bioprocesses.
1999
A. Podgornik, M. Barut, J. Jančar, A. Štrancar, T. Tennikova
Analytical Chemistry, 1999, Vol. 71, No. 15, 2986-2991
High-performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic chromatography of plasmid DNA conformations. However, no study about the separation of small molecules has been performed until now. In this work, we investigated the possibility of gradient and isocratic HPMC of small molecules with Convective Interaction Media disks of different chemistries and tried to explain the mechanism that enables their separation. We demonstrated that it is possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed-phase mode. It was shown that similar peak resolution can be provided in both gradient and isocratic modes.
A. Podgornik, M. Barut, J. Jančar, A. Štrancar
Journal of Chromatography A, 848 (1999) 51–60
In this work, the isocratic separation of oligonucleotides in the ion-exchange mode on thin glycidylmethacrylate–ethylenedimethacrylate (GMA–EDMA) monoliths in the form of commercially available CIM (Convective Interaction Media) disks is presented. It was found that isocratic separation occurs even on monoliths with a thickness of only 0.75 mm. Peak broadening of the components retained on the monolith is proportional to the retention time, which in turn is proportional to the thickness of the monolith. Peak height is inversely proportional to the retention time. From these results it can be concluded that the mechanism of the separation on such monoliths is similar to that in HPLC columns filled with conventional porous particles. The height equivalent to a theoretical plate of GMA–EDMA monoliths is calculated to be 18.0 μm. The capacity factor k′ depends, exponentially, on the salt concentration. The Z factor calculated from fitted equations increases linearly with the oligonucleotide’s length. It was also found that the difference between peak retention volume slightly increases with the flow-rate when the experiments are performed in the range from 0.5 to 7 ml/min. From the similarities between the isocratic separations on conventional columns and on thin GMA–EDMA monoliths it is reasonable to believe that separation based on a multiple adsorption/desorption process also occurs in thin monoliths.