2003

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.

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R. Hahn, E. Berger, K. Pflegerl, A. Jungbauer

Anal. Chem. 2003, 75, 543-548

When small ligands are immobilized onto a porous chromatography medium, only a limited number of binding sites contributes to the interaction with the target molecule. The main part of the ligand molecules is distributed on sites that are not accessible for the target protein due to steric hindrance. To direct the ligand into a well-accessible position, the ligand was conjugated to a large molecule that acted as a placeholder during the immobilization step. Then the placeholder molecule was cleaved off and washed out. Two linear peptides with affinity for lysozyme and human blood coagulation factor VIII, respectively, were studied as model systems. The protected peptide ligand was covalently linked to a 20-kDa poly(ethylene glycol) molecule containing an acid-labile linker. After selective deprotection of the peptide and purification, immobilization of this conjugate on a preactivated chromatography matrix was performed alternatively through the free N-terminus, the ε-amino group of lysine, or the sulfohydryl group of cysteine. After the immobilization reaction, the spacer molecule and remaining protecting groups were cleaved off and the gels were tested by affinity chromatography. This novel immobilization technique substantially increased the binding capacity and the ligand utilization for the target protein, and site-specific immobilization could be demonstrated.

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P. Kramberger, D. Glover, A. Štrancar

American Biotechnology Laboratory, 2003, 21(13), 27-8.

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 M NaOH), 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 article will discuss the columns and their use on human models and plant viruses and pDNA.

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K. Branović, D. Forčič, J. Ivančič, A. Štrancar, M. Barut, T. Kosutic-Gulija, R. Zgorelec, R. Mazuran

Journal of Virological Methods 110 (2003) 163-171

Monolithic chromatography media represent a novel generation of stationary phases introduced in the last 10–15 years providing a chromatography matrix with enhanced mass transfer and hydrodynamic properties. These features allow for an efficient and fast separation of especially large biomolecules like e.g., DNA and viruses. In this study, the enrichment of virus RNA on short monolithic columns prior to molecular detection of viruses is described. Measles and mumps viruses were chosen as model viruses. The results show that it is possible to bind viral RNA on monoliths and concentrate viral nucleic acids from a fairly dilute sample. Consequently, a potential application of short monolithic columns is the concentration of virus RNA to improve the sensitivity and selectivity of viral detection with the possibility of isolating viral RNA from cell-free biological fluids.

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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.

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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.

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A. Štrancar, A. Podgornik, M. Barut, R. Necina

Advances in Biochemical Engineering/ Biotechnology,Vol. 76, 2002

Monolithic supports represent a novel type of stationary phases for liquid and gas chromatography, for capillary electrochromatography, and as supports for bioconversion and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, monolithic supports are cast as continuous homogeneous phases. They represent an approach that provides high rates of mass transfer at lower pressure drops as well as high efficiencies even at elevated flow rates. Therefore, much faster separations are possible and the productivity of chromatographic processes can be increased by at least one order of magnitude as compared to traditional chromatographic columns packed with porous particles. Besides the speed, the nature of the pores allows easy access even in the case of large molecules, which make monolithic supports a method of choice for the separation of nanoparticles like pDNA and viruses. Finally, for the optimal purification of larger biomolecules, the chromatographic column needs to be short. This enhances the speed of the separation process and reduces backpressure, unspecific binding, product degradation and minor changes in the structure of the biomolecule, without sacrificing resolution. Short Monolithic Columns (SMC) were engineered to combine both features and have the potential of becoming the method of choice for the purification of larger biomolecules and nanoparticles on the semi-preparative scale.

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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.

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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.

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K. Pflegerl, R. Hahn, E. Berger, A. Jungbauer

J. Peptide Res., 2002, 59, 174-182

A peptide screened from a combinatorial peptide library with the sequence EYKSWEYC performed best as a ligand for affinity chromatography of human blood coagulation factor VIII (FVIII). With this peptide immobilized on monolithic CIM columns via epoxy groups we were able to capture FVIII from diluted plasma. Rational substitution of amino acids by spot synthesis revealed that lysine and cysteine can be exchanged for almost all other proteinogenic amino acids without loss of affinity to FVIII. This offers the possibility of site-specific attachment via either one of these residues or the N- or C-terminus. The aliphatic positions O5 (tryptophan) and O7 (tyrosine), together with the charged position O6 (glutamic acid), seem to form the core of the binding unit. In the positions with aliphatic amino acids, substitution by tyrosine or phenylalanine, and in the positions with charged amino acids, substitution by aspartic acid or lysine, preserved the affinity to FVIII. The functionality of the selected peptides was confirmed by affinity chromatography. Selective binding and elution could be achieved.

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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.

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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.

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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.

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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.

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L. G. Berruex, R. Freitag

Methods for Affinity-Based Separations of Enzymes and Proteins Methods and Tools in Biosciences and Medicine 2002, 82-114

Macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) (GMAEDM A) disks have been introduced in the late eighties as novel stationary phases for biochromatography, which putatively offer new possibilities, especially for the separation/analysis of large biologicals such as proteins [1]. Various stationary phases based on the GMA-EDM A chemistry are commercially available from BIA Separations d.o.o., Slovenia, under the trade name of CIM® (Convective Interaction Media) disks.

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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.

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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.

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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.

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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.

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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.

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