On May 12th, the biaseparations.com website will be retired and migrated tosartorius.com.Learn moreabout our combined offering today!
2007

P. Brne, A. Podgornik, K. Benčina, B. Gabor, A. Štrancar, M. Peterka

Journal of Chromatography A , 1144 (2007) 120-125

Certain diagnostic, analytical and preparative applications require the separation of immunoglobulin G (IgG) from immunoglobulin M (IgM). In the present work, different ion-exchange methacrylate monoliths were tested for the separation of IgG and IgM. The strong anion-exchange column had the highest dynamic binding capacity reaching more than 20 mg of IgM/ml of support. Additionally, separation of IgM from human serum albumin, a common contaminant in immunoglobulin purification, was achieved on the weak ethylenediamino anion-exchange column, which set the basis for the IgM purification method developed on convective interaction media (CIM) supports. Experiments also confirmed flow independent characteristics of the short monolithic columns.

Purchase full article

Full view

C. K. Zacharis, E. A. Kalaitzantonakis, A. Podgornik, G. Theodoridis

Journal of Chromatography A, 1144 (2007) 126–134

In this study, sequential injection affinity chromatography was used for drug–protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy ΔG, enthalpy ΔH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 × 106 M-1 at pH 7.4 and 39 °C for a single binding site. The associated change in enthalpy (ΔH) was −27.36 kcal mol-1 and the change in entropy (ΔS) was −73 cal mol-1 K-1 for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (λext = 230 nm, λem = 350 nm) and spectrophotometric detection (λ = 280 nm).

Purchase full article

Full view

T. Čerk Petrič, P. Brne, B. Gabor, L. Govednik, M. Barut, A. Štrancar, L. Zupančič Kralj
Journal of Pharmaceutical and Biomedical Analysis 43 (2007) 243–249

In order to enable the detection of low abundance proteins from human plasma, it is necessary to remove high abundance proteins. Among them, human serum albumin and immunoglobulin G represent more than 75% of all such proteins. In this paper, the characterization of short monolithic columns was performed followed by the optimization of a multidimensional approach, known as conjoint liquid chromatography, to deplete human serum albumin and immunoglobulin G from a human plasma sample. Two different chromatographic modes were used: ion-exchange chromatography and affinity chromatography. A monolithic stationary phase (convective interaction media disk) bearing strong anion-exchange groups and another immobilized with protein G were placed in series into one housing. The optimal binding conditions were found that removed a majority of human serum albumin and immunoglobulin G from the human plasma sample. This method was compared to the depletion using a combination of pseudo-affinity and affinity columns. The results of the human serum albumin and immunoglobulin G depletion were confirmed by 2D electrophoresis. It has been shown that anion-exchange and affinity chromatography using convective interaction media monolithic columns can represent an efficient complementary technique for human serum albumin and immunoglobulin G removal from human plasma.

Purchase full article

Full view

2005

Y.-P. Lim, D. Josić, H. Callanan, J. Brown, D. C. Hixson

Journal of Chromatography A, 1065 (2005) 39–43(2005) 39–43

Epoxy-activated monolithic CIM disks seem to be excellent supports for immobilization of protein ligands. The potential use of enzymes, immobilized on monolithic disks for rapid preparative cleavage proteins in solution was investigated. Digestion of complex plasma proteins was demonstrated by using inter-alpha inhibitors with elastase, immobilized on epoxy-activated CIM disks. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAb 69.31) was developed. MAb 69.31 blocks the inhibitory activity of inter-alpha inhibitor proteins to serine proteases. These results suggest that the epitope defined by this antibody is located within or proximal to the active site of the inhibitor molecule. This antibody, immobilized on monolithic disk, was used for very rapid isolation of inter-alpha proteins. The isolated complex protein was used for enzymatic digestion and isolation of cleavage products, especially from inter-alpha inhibitor light chain to elucidate precisely the target sequence for MAb 69.31 by N-terminal amino acid sequencing. Bovine pancreatic elastase immobilized on monolithic disk cleaves inter-alpha inhibitor protein complex into small fragments which are still reactive with MAb 69.31. One of these proteolytic fragments was isolated and partially sequenced. It could be shown that this sequence is located at the beginning of two proteinase inhibitor domains of the inter-alpha inhibitor light chain (bikunin). Elastase immobilized on monolithic disk offers a simple and rapid method for preparative isolation of protease cleavage fragments. The immobilized enzyme is stable and still active after repeated runs. A partial or complete digestion can be achieved by varying the flow rate.

Purchase full article

Call

Send SMS

Add to Skype

You'll need Skype CreditFree via Skype

Full view

2004

T. Hall, D. C. Wood, C. E. Smith

Journal of Chromatography A, 1041 (2004) 87–93(2004) 87–93

Monolithic media were compared with Q- and SP-Sepharose high performance chromatography for preparative purification and with Q- and SP-5PW chromatography for analysis of a pegylated form of myelopoietin (MPO), an engineered hematopoietic growth factor. The use of either monolithic or Sepharose based supports for preparative chromatography produced highly purified pegylated MPO with the monolithic media demonstrating peak resolution and repeatability at flow rates of 1 and 5 ml/min resulting in run times as much as five-fold shorter compared to Sepharose separations. The monolithic disks also resulted in 10-fold shorter run times for the analytical chromatography, however, their chromatographic profiles and peak symmetry were not as sharp compared to their Q-5PW and SP-5PW counterparts.

Purchase full article

Full view

E. Vlakh, A. Novikov, G. Vlasov, T. Tennikova

Journal of Peptide Science, 10: 719–730 (2004)

Monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) can be used directly as sorbents for affinity chromatography after solid phase peptide synthesis. The quality of the synthesized products, the amount of grown peptides on a support and the reproducibility of the process must be considered. A determination of the quantity of the introducing β-Ala (and, consequently, the total amount of synthesized peptide) was carried out. Three peptides complementary to recombinant tissue plasminogen activator (t-PA) have been synthesized using Fmoc-chemistry on GMA-EDMA disks. The peptidyl ligands were analysed by amino acid analysis, ES-MS and HPLC methods.

The affinity binding parameters were obtained from frontal elution data. The results were compared with those established for GMA-EDMA affinity sorbents formed by the immobilization of the same but separately synthesized and purified ligands. The immobilization on GMA-EDMA disks was realized using a one-step reaction between the amino groups of the synthetic ligand and the original epoxy groups of monolithic material. The affinity constants found for two kinds of sorbent did not vary significantly. Finally, the directly obtained affinity sorbents were tested for t-PA separation from a cellular supernatant.

Purchase full article

Full view

D. Ren, N. A. Penner, B. E. Slentz, H. D. Inerowicz, M. Rybalko, F. E. Regnier

Journal of Chromatography A, 1031 (2004) 87–92(2004) 87–92

Immobilized copper(II) affinity chromatography [Cu(II)-immobilized metal affinity chromatography (IMAC)] has been used in proteomics to simplify sample mixtures by selecting histidine-containing peptides from proteolytic digests. This paper examines the specificity of four different support materials with an iminodiacetic acid (IDA) stationary phase in the selection of only histidine-containing peptides in the single step capture-release mode. Three of the sorbents examined were commercially available: HiTrap Chelating HP (agarose), TSK Chelate-5PW, and Poros 20MC. IDA was also immobilized on CIM discs (monolithic glycidylmethacrylate-ethylene dimethacrylate). Tryptic digests of transferrin and β-galactosidase were used as model samples to evaluate these sorbents. It was found that among the examined matrices, the TSK Chelate-5PW sorbent bound histidine-containing peptides the strongest, while Poros matrix was found to have a high degree of non-specific bindings. Agarose-based columns showed relatively high selectivity and specificity.

Purchase full article

Full view

E. G. Vlakh, A. Tappe, C. Kasper, T. B. Tennikova

Journal of Chromatography B, 810 (2004) 15–23

Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.

Purchase full article

Full view

E. G. Vlakh, A. Tappe, C. Kasper, T. B. Tennikova

Journal of Chromatography B, 810 (2004) 15–23

Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.

Purchase full article

Full view

E. G. Vlakh, A. Tappe, C. Kasper, T. B. Tennikova

Journal of Chromatography B, 810 (2004) 15–23

Plasminogen activators are the proteases which convert plasminogen into plasmin dissolving, in its turn, the major component of blood clots, fibrin. They are extremely useful in heart attack therapy. Modern and most appropriate way of scaled up production of these valuable proteins is gene engineering. In this case, a separation and a purification of target product become the important steps of the whole process. Recently developed affinity chromatography on short monolithic columns seems to be a very attractive method for these purposes. High speed of a process prevents the protein’s denaturation due to temperature or/and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows considering only biospecific complexing as time limiting step. Specificity of several synthetic peptides to plasminogen activators have been studied by affinity chromatography on short monolithic columns. Peptide ligands were synthesized by conventional solid phase peptide synthesis (SPPS). The immobilization procedure was carried out as a one step process at static conditions. The results of quantitative evaluation of such affinity interactions were compared with those established for plasminogen that is the natural affinity counterpart to both proteases. Additionally, some of investigated peptides were synthesized directly on GMA–EDMA disks and their affinity properties were compared with those established for the case of immobilized ligands. The possibility of using of synthetic peptidyl ligands for plasminogen activators isolation from native cell supernatant and model protein mixtures has been demonstrated.

Purchase full article

Full view

E. Vlakh, N. Ostryanina, A. Jungbauer, T. Tennikova

Journal of Biotechnology 107 (2004) 275–284

Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM® Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM® affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.

Purchase full article

Full view

P. N. Nesterenko, M. A. Rybalko

Mendeleev Commun. 2004

The continuous flow gradient and its effect on chromatographic parameters were investigated for the separations of inorganic anions on a monolithic porous disk with bonded hydroxyproline residues.

Purchase full article

Full view

2003

E. G. Vlakh, G. A. Platonova, G. P. Vlasov, C. Kasper, A. Tappe, G. Kretzmer, T. B. Tennikova

Journal of Chromatography A, 992 (2003) 109–119

The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-l-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.

Purchase full article

Full view

K. Branović, A. Buchacher, M. Barut, A. Štrancar, D. Josić

Journal of Chromatography B, 790 (2003) 175–182

It has been shown in a previous study that monolithic columns can be used for downstream processing of different concentrates of clotting factor IX [K. Branović et al., J. Chromatogr. A 903 (2000) 21]. This paper demonstrates that such supports are useful tools also at an early stage of the purification process of factor IX from human plasma. Starting with the eluate after solid-phase extraction with DEAE-Sephadex, the use of monolithic columns has allowed much better purification than that achieved with conventional anion-exchange supports. The period of time required for separation is also much reduced. In up-scaling experiments, separations are carried out with 8, 80 and 500 ml columns. A volume of 1830 ml of DEAE-Sephadex eluate, containing a total of 27.6 g of protein and 48.500 IU of factor IX is applied to the 500 ml monolithic column. This corresponds to a separation on a pilot scale. The results of this separation after up-scaling are comparable to those obtained with the 8 ml column on a laboratory scale.

Purchase full article

Full view

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.

Purchase full article

Full view

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.

Purchase full article

Full view

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.

Purchase full article

Full view

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.

Purchase full article

Full view

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.

Purchase full article

Full view

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.

Purchase full article

Full view