Publications
2002
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. Branović, G. Lattner, M. Barut, A. Štrancar, D. Josić, A. Buchacher
Journal of Immunological Methods 9211 (2002) 20;271(1-2):47-58
Transferrin and albumin are often present in immunoglobulin G (IgG) concentrates and are considered as impurities. Therefore, it is important to determine their concentration in order to obtain a well-characterized biological product. Here, we describe their determination based on conjoint liquid chromatography (CLC). The established method combines two different chromatographic modes in one step: affinity and ion-exchange chromatography (IEC) combined in one column. Therefore, two CIM Protein G and one CIM quaternary amine (QA) monolithic disks were placed in series in one housing forming a CLC monolithic column. Binding conditions were optimized in a way that immunoglobulins were captured on the CIM Protein G disks, while transferrin and albumin were bound on the CIM QA disks. Subsequently, transferrin and albumin were eluted separately by a stepwise gradient with sodium chloride, whereas immunoglobulins were released from the Protein G ligands by applying low pH. A complete separation of all three proteins was achieved in less than 5 min. The method permits the quantification of albumin and transferrin in IgG concentrates and has been successfully validated.
T. V. Gupalova, O. V. Lojkina, V. G. Palagnuk, A. A. Totolian, T.B. Tennikova
Journal of Chromatography A, 949 (2002) 185–193
The recombinantly produced different forms of protein G, namely monofunctional immunoglobulin G (IgG) binding, monofunctional serum albumin (SA) binding and bifunctional IgG/SA binding proteins G, are compared with respect to their specific affinities to blood IgG and SA. The affinity mode of the recently developed high-performance monolithic disk chromatography has been used for fast quantitative investigations. Using single affinity disks as well as two discs stacked into one separation unit, one order of magnitude in adsorption capacities for IgG and SA were found both for monofunctional and bifunctional protein G forms used as specific affinity ligands. However, despite the adsorption difference observed, the measured dissociation constants of the affinity complexes seemed to be very close. The analytical procedure developed can be realized within a couple of minutes. Up-scaling of the developed technology was carried out using another type of monolithic materials, i.e. CIM® affinity tubes.
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.
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.
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.
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.
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.
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.
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.