Publications
2008
J. Ivancic-Jelecki, M. Brgles, M. Šantak, D. Forčić
Journal of Chromatography A 1216 (2009) 2717-2724
Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a Sartorius BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.
2007
E. Müller, C. Mann
Journal of Chromatography A, 1144 (2007) 30-39(2007) 30-39
The electro-acoustic effects, namely the ion vibration potential (IVP) and the colloidal vibration current (CVI), colloidal vibration potential (CVP) first described by P. Debye [P. Debye, J. Chem. Phys. 1 (1933) 13], are a result of charge separation of bound or free ions at different degrees by ultrasonic waves. Today commercial instruments are available to investigate liquid homogeneous and heterogeneous systems. In the present paper the application of this technique for the characterization of salts, protein solutions and resins for biochromatography is shown and valuable information about resins can be derived in a short time. Various resins were investigated with the following results: (1) the CVI magnitude is dependent of several parameters (such as particle size distribution, volume fraction, density difference); (2) the CVI is influenced by the surface modification of the resins. Polymeric modifications decrease the value of CVI. The CVI is generally lower for high capacity resins; (3) the measurement of the electro-acoustic effects can be used to detect small changes in resins. The CVI is dependent of the amount of adsorbed protein in “native” and denatured state.
I. V. Kalashnikova, N. D. Ivanova, T. G. Evseeva, A. Yu. Menshikova, E. G. Vlakh, T. B. Tennikova
Journal of Chromatography A, 1144 (2007) 40–47(2007) 40–47
The subject of this paper is an investigation of the peculiarities of dynamic adsorption behavior of nanoparticles. For this purpose, virus-mimicking synthetic particles bearing different proteins at their outer surface were specially constructed using two approaches, e.g. the cross-linking of proteins and modification of polystyrene microsphere surface by proteins. Two chromatographic modes, namely ion-exchange and affinity liquid chromatography on ultra-short monolithic columns [Convective Interaction Media (CIM) DEAE and CIM QA disks] have been used as a tool for dynamic adsorption experiments. Such parameters as maximum adsorption capacity and its dependence on applied flow rate were established and compared with those obtained for individual proteins. Similarly to individual proteins, it was shown that the maximum of adsorption capacity was not changed at different flow rates. In addition, the permeability of porous space of used monolithic sorbents appeared to be sufficient for efficient separation of large particles and quite similar to the well-studied process applied for individual proteins.
N. Delmotte, U. Kobold, T. Meier, A. Gallusser, A. Strancar, C. G. Huber
Anal Bioanal Chem (2007) 389:1065–1074
Immunoadsorbers based on 2.0 × 6.0 mm i.d., epoxy-bearing, methacrylate-based monolithic disks were developed in order to target myoglobin and N-terminal pro-natriuretic peptide (NT-proBNP), two biomarkers involved in cardiovascular disease. In both cases, antibodies were successfully coupled to the polymeric disk material. The developed immunoadsorbers permitted the selective isolation of myoglobin and NT-proBNP from human serum. Myoglobin was successfully isolated and detected from serum samples at concentrations down to 250 fmol μL-1. However, the affinity of the antibodies was not sufficient for the analysis of low-concentration clinical samples. Frontal analysis of anti-NT-proBNP disks revealed the ability of the immunoadsorber to bind up to 250 pmol NT-proBNP, which is more than sufficient for the analysis of clinical samples. Anti-NT-proBNP disks showed good stability over more than 18 months and excellent batch-to-batch reproducibility. Moreover, anti-NT-proBNP disks permitted the isolation of NT-proBNP at concentrations down to 750 amol μL−1 in serum, corresponding to concentrations of strongly diseased patients. Using reversed-phase trapping columns, the detection of NT-proBNP eluted from immunoadsorbers by mass spectrometry was achieved for concentrations down to 7.8 fmol μL-1.
D. Josić, J. G. Clifton
Journal of Chromatography A, 1144 (2007) 2-13
An overview on the utilization of monoliths in proteomics technology will be given. Both silica- and polymer-based monoliths have broad use for microseparation of tryptic peptides in reversed-phase (RP) mode before identification by mass spectrometry (MS) or by MS/MS. For two-dimensional (2D) LC separation of peptides before MS or MS/MS analysis, a combination of ion-exchange, usually cation-exchange (CEX) chromatography with RP chromatography on monolithic supports can be employed. Immobilized metal ion affinity chromatography monoliths with immobilized Fe3+-ions are used for the isolation of phosphopeptides. Monoliths with immobilized affinity ligands are usually applied to the rapid separation of proteins and peptides. Miniaturized reactors with immobilized proteolytic enzymes are utilized for rapid on- or offline digestion of isolated proteins or protein mixtures prior to identification by LC–MS/MS. Monoliths also have broad potential for application in sample preparation, prior to further proteomic analyses. Monolithic supports with large pore sizes can be exploited for the isolation of nanoparticles, such as cells, organelles, viruses and protein aggregates. The potential for further adoption of monolithic supports in protein separation and enrichment of low abundance proteins prior to proteolytic digestion and final LC–MS/MS protein identification will be discussed.
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.
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).
K. Benčina, M. Benčina, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1160 (2007) 176–183
The chromatography of mechanically sensitive macromolecules still represents a challenge. While larger pores can reduce the mechanically induced cleavage of large macromolecules and column clogging, the column performance inevitably decreases. To investigate the effect of pore size on the mechanical degradation of DNA, column permeability and enzyme biological activity, methacrylate monoliths with different pore sizes were tested. Monolith with a 143 nm pore radius mechanically damaged the DNA and was clogged at flow rates above 0.5 ml min−1 (26 cm h−1). For monoliths with a pore radius of 634 nm and 2900 nm, no mechanical degradation of DNA was observed up to 5 ml min−1 (265 cm h−1) above which the HPLC itself became the main source of damage. A decrease of a permeability appeared at flow rate 1.8 ml min−1 (95 cm h−1) and 2.3 ml min−1 (122 cm h−1), respectively. The effect of the pore size on enzyme biological activity was tested with immobilized DNase and trypsin on all three monoliths. Although the highest amount of enzyme was immobilized on the monolith with the smallest pores, monolith with the pore radius 634 nm exhibited the highest DNase biological activity probably due to restricted access for DNA molecules into the small pores. Interestingly, specific biological activity was increasing with a pore size decrease. This was attributed to higher number of contacts between a substrate and immobilized ligand.
S. Yamamoto, M. Nakamura, C. Tarmann, A. Jungbauer
Journal of chromatography 1144 (2007) 155-160
Linear gradient elution experiments were carried out on monolithic anion-exchange chromatography (AEC) with oligo-DNAs of various sizes (4–50mer, molecular weight MW = 1200–15,000) and compositions in order to investigate the retention mechanism. The binding site (B) values as well as the peak salt elution concentration IR values were determined. The B values determined for the monolithic AEC were similar to the values for non-porous AEC and porous AEC. The B value increased linearly with the number of charges (bases) of single-strand DNA when MW is less than ca. 3600 (12mer). When MW is greater than 6000, the slope of B versus MW decreased, and became very small at MW > 30,000. The IR value also increased linearly with MW for MW < 6000, and slightly with MW for MW > 10,000. It was shown that a very difficult separation of a single-strand 50mer poly(T) and a double-strand 50mer poly(A) and poly(T) was accomplished within 10 min by using a very shallow gradient at a high initial salt concentration (0.5 M) and a high flow-velocity (2.7 cm/min).
M. Brgles, B Halassy, J. Tomašić, M. Šantak, D. Forčić, M. Barut, A. Štrancar
Journal of Chromatography A 1144 (2007) 150-154
A high-performance liquid chromatography (HPLC) method for the determination of DNA entrapment efficiency in liposomes has been developed. Plasmid DNA was encapsulated into positively charged liposomes. Non-entrapped DNA was separated by ultracentrifugation from liposomes and supernatant was chromatographed on Convective Interaction Media (CIM) DEAE disk. The elution of DNA was monitored by the absorbance at 260 nm and the quantity of DNA in the tested sample was calculated from the integrated peak areas using the appropriate standard curve. This method is fast, simple, precise and does not require any kind of DNA labelling in contrast with mostly used methods for determination of DNA entrapment efficiency.
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.
2006
M. Krajačić, J. Ivancic-Jelecki, D. Forčić, A. Vrdoljak, D. Škorić
Journal of Chromatography A, 1144 (2007) 111-119
Replicative double-stranded RNA (dsRNA) is useful in preliminary identification of Cucumber mosaic virus and its satellite RNA (satRNA). This plant pathogen complex yields sufficient quantity of the replicative RNA form that can be isolated by chromatography on chemically unmodified graded cellulose powder (CF-11). In this work, much faster and more efficient procedure using DEAE monoliths was developed in which dsRNA was separated from other species in total nucleic acids extract originating from the infected plant tissue. The developed chromatographic method revealed the pathogens’ presence in only 15 min, avoiding nucleic acid precipitation and electrophoretic analysis.
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.
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D. Forčić, K. Branovič Čakanič, J. Ivančič, R. Jug, M. Barut, A. Štrancar, R. Mazuran
Analytical Biochemistry 336 (2005) 273-278
Analysis of crude samples from biotechnological processes is often required to demonstrate that residual host cell impurities are reduced or eliminated during purification. Current knowledge suggests that a continuous-cell-line DNA can be considered a cellular contaminant rather than a significant risk factor requiring removal to extremely low levels. Anion-exchange chromatography is one of the most important methods used in the downstream processing and analysis of different biomolecules. In this article, an application using Convective Interaction Media monolithic columns to improve the detection of residual cellular DNA is described.
S. Jerman, A. Podgornik, K. Cankar, N. Čadež, M. Skrt, J. Žel, P. Raspor
Journal of Chromatography A 1065 (2005) 107-113
The availability of sufficient quantities of DNA of adequate quality is crucial in polymerase chain reaction (PCR)-based methods for genetically modified food detection. In this work, the suitability of anion-exchange CIM (Convective Interaction Media; Sartorius BIA Separations, Ljubljana, Slovenia) monolithic columns for isolation of DNA from food was studied. Maize and its derivates corn meal and thermally pre-treated corn meal were chosen as model food. Two commercially available CIM disk columns were tested: DEAE (diethylaminoethyl) and QA (quaternary amine). Preliminary separations were performed with standard solution of salmon DNA at different pH values and different NaCl concentrations in mobile phase. DEAE groups and pH 8 were chosen for further isolations of DNA from a complex matrix—food extract. The quality and quantity of isolated DNA were tested on agarose gel electrophoresis, with UV-scanning spectrophotometry, and by amplification with real-time PCR. DNA isolated in this way was of suitable quality for further PCR analyses. The described method is also applicable for DNA isolation from processed foods with decreased DNA content. Furthermore, it is more effective and less time-consuming in comparison with the existing proposed methods for isolation of DNA from plant-derived foods.
D. Forčić, K. Branović-Čakanić, J. Ivančić, R. Jug, M. Barut, A. Štrancar
Journal of Chromatography A 1065 (2005) 115-120
The isolation and purification of nucleic acids is essential for many procedures in molecular biology. After showing that bacterial and eukaryotic genomic DNA can be specifically bound to the CIM DEAE monolithic column, this characteristic was exploited in development of a simple and fast chromatographic procedure for isolation and purification of genomic DNA from cell lysates that does not include the usage of toxic organic solutions. The purity and the quality of the isolate as well as the duration of the procedure was similar to other chromatographic methods used today for isolation of genomic DNA, but the initial sample volume was not restricted.
J. Urthaler, R. Schlegl, A. Podgornik, A. Štrancar, A. Jungbauer, R. Necina
Journal of Chromatography A 1065 (2005) 93-106
The demand of high-purity plasmid DNA (pDNA) for gene-therapy and genetic vaccination is still increasing. For the large scale production of pharmaceutical grade plasmids generic and economic purification processes are needed. Most of the current processes for pDNA production use at least one chromatography step, which always constitutes as the key-step in the purification sequence. Monolithic chromatographic supports are an alternative to conventional supports due to their excellent mass transfer properties and their high binding capacity for pDNA. Anion-exchange chromatography is the most popular chromatography method for plasmid separation, since polynucleotides are negatively charged independent of the buffer conditions. For the implementation of a monolith-based anion exchange step into a pDNA purification process detailed screening experiments were performed. These studies included supports, ligand-types and ligand-densities and optimization of resolution and productivity. For this purpose model plasmids with a size of 4.3 and 6.9 kilo base pairs (kbp) were used. It could be shown, that up-scaling to the production scale using 800 ml CIM Convective Interaction Media radial flow monoliths is possible under low pressure conditions. CIM DEAE was successfully implemented as intermediate step of the cGMP pDNA manufacturing process. Starting from 200 l fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 l tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.
M. Peterka, P. Kramberger, A. Štrancar
WANG, Perry G. (ur.). Monolithic chromatography and its modern applications. St Albans: ILM publications, 2010, pg. 489-508
Downstream processing (DSP) for purification can become a significant bottleneck in the production of novel biotherapeutics, such as viral vectors and vaccines (viral or DNA). Although different techniques can be used for the purification of large molecules and particles, liquid chromatography is the preferred method as it achieves the purity required by regulatory agencies. Despite the popularity of conventional chromatographic media, the diffusional mass transfer of large molecules and relatively small pore size remain limiting factors for the efficient separation of large biomolecules and particles. Methacrylate monoliths are a single-piece chromatographic support that consists of a highly porous material with an interconnected network of channels. The transport mechanism is predominantly based on convection, which allows rapid mass transfer between the mobile and stationary phase and so results in short separation times. Additionally, most of the active sites are located in the open, large channel structure and are therefore easily accessible, which results in a high DBC (DBC) for large molecules and viral particles. These characteristics make methacrylate monoliths an ideal chromatographic support for the separation and purification of extremely large molecules, such as large proteins, different types of DNA and virus particles.
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.
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.