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2002

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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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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2001

P. Svete, R. Milačič, B. Mitrović, B. Pihlar

The Royal Society of Chemistry 2001, Analyst, 2001, 126, 1346–1354

Analytical procedures were developed for the speciation of Zn using fast protein liquid chromatography (FPLC), flame atomic absorption spectrometry (FAAS) and convective interaction media (CIM) fast monolithic chromatography with FAAS and electrospray (ES)-MS-MS detection. The investigation was performed on synthetic solutions (2 µg cm-3 Zn) of hydrated Zn2+ species and Zn complexes with citrate, oxalate and EDTA (ligand-to-Zn molar ratio 100 : 1) over a pH range from 5.4 to 7.4. It was found that Zn interacts with various buffers and the careful adjustment of the pH with diluted solutions of KOH is, therefore, required. FPLC separations were carried out on a Mono Q HR 5/5 strong anion-exchange column, applying an aqueous 1 mol dm-3 NH4NO3 linear gradient elution over 15 min, at a flow rate of 1.0 cm3 min−1. The separated Zn species were determined in 1.0 cm3 eluate fractions “off line” by FAAS. Speciation of Zn was also performed on a weak anion-exchange CIM DEAE fast monolithic disc by applying an aqueous 0.4 mol dm-3 NH4NO3 linear gradient elution over 7.5 min, at a flow rate of 2.0 cm3 min−1 and determination of the separated Zn species in 1.0 cm3 eluate fractions “off line” by FAAS. Zn-binding ligands in separated fractions were also characterized by electrospray (ES)-MS-MS analysis. The CIM DEAE disc was found to be more efficient in the separation of negatively charged Zn complexes than the Mono Q FPLC column. On the CIM DEAE disc Zn–citrate was separated from both Zn–oxalate and from Zn–EDTA. All these species were also separated from hydrated Zn2+, which was eluted with the solvent front. This method has an advantage over commonly used analytical techniques for the speciation of Zn which are only able to distinguish between labile and strong Zn complexes. Good repeatability of the measurements (RSD 2–4%), tested for six parallel determinations (2 µg cm-3 Zn) of Zn–EDTA, Zn–citrate and Zn–oxalate was found at a pH of 6.4 on a CIM DAEA disc. The limit of detection (3s) for the separated Zn species was 10 ng cm-3. The proposed analytical procedure was applied to the speciation of Zn in aqueous soil extracts and industrial waste water from a lead and zinc mining area.

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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.

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2000

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.

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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.

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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.

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1998

C. Kasper, L. Meringova, R. Freitag, T. Tennikova

Journal of Chromatography A, 798 (1998) 65-72

A fast affinity method for the semi-preparative isolation of recombinant Protein G from E. coli cell lysate is proposed. Rigid, macroporous affinity discs based on a glycidyl methacrylate–co-ethylene dimethacrylate polymer were used as chromatographic supports. The specific ligands (here human immunoglobulin G, hIgG) were immobilized by the one-step reaction between native epoxy groups of the polymer surface and ϵ-amino groups of the IgG molecules. No intermediate spacer was necessary to reach full biological activity of the ligand. The globular affinity ligands are located directly on the pore wall surface and are thereby freely accessible to target molecules (here Protein G) migrating with the mobile phase through the pores. It is shown that the conditions chosen for the hIgG immobilization do not involve an active site of the protein and thus do not bias the formation of the affinity complex. Chromatographically determined constants of dissociation of hIgG–Protein G affinity complexes confirm the high selectivity of this separation method. Two different aspects of the affinity separation are discussed, which differ mostly in terms of scale. In disc chromatography, high volumetric flow velocities are possible because of the small backpressure. Since in addition the mass transfer is more efficient, it becomes possible to achieve very short analysis times. The discs proposed can be used in a single-step enrichment of Protein G from lysates of non-pathogenic E. coli. Gel electrophoresis data are used to demonstrate the high degree of purity achieved for the final product.

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D. Josić, H. Schwinn, A. Štrancar, A. Podgornik, M. Barut, Y. P. Lim, M. Vodopivec

Journal of Chromatography A, 803 (1998) 61–71

Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase IV, which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate–polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.

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C. Kasper, L. Meringova, R. Freitag, T. Tennikova

Journal of Chromatography A, 798 (1998) 65-72

A fast affinity method for the semi-preparative isolation of recombinant Protein G from E. coli cell lysate is proposed. Rigid, macroporous affinity discs based on a glycidyl methacrylate–co-ethylene dimethacrylate polymer were used as chromatographic supports. The specific ligands (here human immunoglobulin G, hIgG) were immobilized by the one-step reaction between native epoxy groups of the polymer surface and ϵ-amino groups of the IgG molecules. No intermediate spacer was necessary to reach full biological activity of the ligand. The globular affinity ligands are located directly on the pore wall surface and are thereby freely accessible to target molecules (here Protein G) migrating with the mobile phase through the pores. It is shown that the conditions chosen for the hIgG immobilization do not involve an active site of the protein and thus do not bias the formation of the affinity complex. Chromatographically determined constants of dissociation of hIgG–Protein G affinity complexes confirm the high selectivity of this separation method. Two different aspects of the affinity separation are discussed, which differ mostly in terms of scale. In disc chromatography, high volumetric flow velocities are possible because of the small backpressure. Since in addition the mass transfer is more efficient, it becomes possible to achieve very short analysis times. The discs proposed can be used in a single-step enrichment of Protein G from lysates of non-pathogenic E. coli. Gel electrophoresis data are used to demonstrate the high degree of purity achieved for the final product.

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1997

A. Štrancar, M. Barut, A. Podgornik, P. Koselj, H. Schwinn, P. Raspor, D. Josić

Journal of Chromatography A, 760 (1997) 117-123

Membranes as well as compact porous disks are successfully used for fast analytical separations of biopolymers. So far, technical difficulties have prevented the proper scaling-up of the processes and the use of membranes and compact disks for preparative separations in a large scale. In this paper, the use of a compact porous tube for fast preparative separations of proteins is shown as a possible solution to these problems. The units have yielded good results, in terms of performance and speed of separation. The application of compact porous tubes for the preparative isolation of clotting factor VIII from human plasma shows that this method can even be used for the separation of very sensitive biopolymers. As far as yield and purity of the isolated proteins are concerned, the method was comparable to preparative column chromatography. The period of time required for separation was five times shorter than with corresponding column chromatographic methods. Compact porous disks made of the same support material can also be used for in-process analysis in order to control the separation. The quick response, which is obtained from these units within 5 to 60 s, allows close monitoring of the purification process.

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1996

N.I. Dubinina, O.I. Kurenbin, T.B. Tennikova

Journal of Chromatography A, 753 (1996) 217-225

Since the influence of column length on protein resolution in high-performance liquid chromatography (HPLC) is not clear, different viewpoints presented in the literature are analysed in detail. The influence of gradient steepness on the length of the working column part (X0) or the part of a column in which the quasi-steady state is attained was studied. The equation for estimating the X0 value was obtained for the general case of the retention model. It was shown that at steep gradients only a short part of the column is used as the working part on which all separation processes develop. The other part of a column is a ballast where the protein zone migrates in a regime of parallel transfer. These results form a theoretical basis for high-performance membrane chromatography. As was shown experimentally, this method makes it possible to perform protein separation at low gradient times with appropriate resolution, comparable with that of HPLC.

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1994

D. Josić, Y.P. Lim, A. Štrancar, W. Reutter

Journal of Chromatography B, 662 (1994) 217-226

The separation of annexins, calcium-binding plasma membrane-associated proteins from rat liver and Morris hepatoma 7777 by high-performance membrane chromatography (HPMC) is described. The annexins with low molecular masses, CBP 33 and CBP 35, and the annexin with a high molecular mass, CBP 65/67, can be separated within 10 min from one another by anion-exchange HPMC under non-denaturing conditions. The separation devices used consist of compact, porous disks (QuickDisk) on the one hand and of bundled membranes made of cellulose fibers (MemSep) on the other. Both have been found to be equally well suited for this separation. The annexins obtained in this way are subsequently bound to epoxy-activated porons disks and used for the separation of monospecific polyclonal antibodies against the annexin CBP 65/67.

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1992

D. Josić, J. Rauch, K. Löster, O. Baum, W. Reutter

Journal of Chromatography A, 590 (1992) 59-76

Porous discs made of poly(glycidyl methacrylate) were used for high-performance membrane chromatography (HPMC) of proteins. In model experiments, separations of standard proteins by anion-exchange HPMC using a DEAE disc were carried out. The influences of sample distribution and disc diameter and thickness on separation performance were studied. The separation disc allowed a scaling-up from analytical (diameter 10 mm) to semi-preparative (diameter 50 mm) dimensions. In an application study, separations with anion-exchange and affinity HPMC were carried out using different complex samples such as rat serum and plasma membrane proteins. In all experiments the results on poly(glycidyl methacrylate) discs were comparable to those achieved on adequate high-performance liquid chromatographic (HPLC) columns. However, the separations on HPMC discs could be carried out faster than corresponding separations on HPLC columns. The pressure drop on the discs was low even at high flow-rates. The experiments show that the poly(glycidyl methacrylate) discs used are especially suitable for the isolation of proteins and other biopolymers which occur in a diluted state in complex mixtures.

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1990

T. B. Tennikova, B. G. Belenkii, F. Švec

Journal of Liquid Chromatography, 13(1), 63-70 (1990)

Basing on the fact that only short layers of a chromatographic column contribute to the separation in the interaction chromatography, 1 mm thick membranes from macroporous methacrylate polymer provided with functional groups were synthetized and used for protein separation. The chromatograms show that the separation is fully comparable with that experienced on a filled column but the advantage of a membrane is up to two orders of magnitude lower pressure during the process and very high loading reaching up to 40 g/m2. This recommends the high performance membrane chromatography also for large scale preparative separations.

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