Publications
2005
N. Lendero, J. Vidič, P. Brne, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1065 (2005) 29-38(2005) 29 - 38
The objective of this study was to develop a fast, simple, non-destructive, non-toxic and low-priced method for determining the amount of ionic groups on resins, since the conventional titration method fails to give proper results on methacrylate monoliths. After the column had been pre-saturated with a high concentration buffer solution, a low concentration buffer solution of the same pH value was pumped through the column. Measuring pH and absorbance, the profiles with a shape of typical break-through curve were obtained. It was shown that the time of the pH transient, which appeared under such conditions, could be used as a measure of the total ionic capacity of ion-exchange monolithic columns. The effect of the column length, linear velocity and varying concentrations of buffer solutions on the time of the pH transient was examined. The method was shown to be suitable for determining the amount of ionic groups on both anion and cation monolithic columns. In addition, it could also be applied to particle bed columns. The time of the pH transient and the protein dynamic binding capacity were also compared and it was concluded that for a given monolith the protein capacity can be derived from the data obtained by the new method.
I. Mihelič, D. Nemec, A. Podgornik, T. Koloini
Journal of Chromatography A, 1065 (2005) 59-67(2005) 59 - 67
Pressure drop analysis in commercial CIM disk monolithic columns is presented. Experimental measurements of pressure drop are compared to hydrodynamic models usually employed for prediction of pressure drop in packed beds, e.g. free surface model and capillary model applying hydraulic radius concept. However, the comparison between pressure drop in monolith and adequate packed bed give unexpected results. Pressure drop in a CIM disk monolithic column is approximately 50% lower than in an adequate packed bed of spheres having the same hydraulic radius as CIM disk monolith; meaning they both have the same porosity and the same specific surface area. This phenomenon seems to be a consequence of the monolithic porous structure which is quite different in terms of the pore size distribution and parallel pore nonuniformity compared to the one in conventional packed beds. The number of self-similar levels for the CIM monoliths was estimated to be between 1.03 and 2.75.
T. B. Tennikova, J. Reusch
Journal of Chromatography A, 1065 (2005) 13-17(2005) 13 - 17
The history of the development of short monolithic beds is described.
J. Vidić, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1065 (2005) 51-58(2005) 51-58
The influence of glass surface modification in order to determine strength of the monolith attachment was studied. Modification consists of pre-treatment of the glass with chemicals or boiling in deionized water, silanization and drying has been investigated on different types of glass. Amount of silane groups was determined by measurement of the contact angle between the glass surface and water drop. The highest values were found for soda–lime glass. Strength of the monolith attachment was established by pumping ethanol through the monolithic capillaries and measuring the pressure drop at which monolith was dislodged. Surprisingly, it was found that the critical part of the glass surface modification procedure is glass pre-treatment. Good results were obtained with glass boiled in water for 2.5 h or more.
S. Yamamoto, A. Kita
Journal of Chromatography A, 1065 (2005) 45-50(2005) 45-50
Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.
K. Isobe, Y. Kawakami
Journal of Chromatography A, 1065 (2005) 129-134
Chromatography conditions for two types of convection interaction media (CIM) tube monolithic column, DEAE-8 and C4-8, were investigated using three enzymes from different microorganisms. The enzymes were adsorbed on a CIM DEAE-8 tube column under the same conditions as conventional DEAE columns. The CIM C4-8 tube column required a high concentration of ammonium sulfate compared to the conventional C4 column for adsorbing the enzymes. The separation of enzymes on the CIM tube column chromatography was not affected at flow rates between 0.15 and 1.25 volumes of the column per min. Both columns were successfully applied to the purification of enzymes from crude enzyme solution. Thus, both CIM tube monolithic columns proved useful in greatly reducing the purification time, and could be used at any stage of enzyme purification.
I. Vovk, B. Simonovska, M. Benčina
Journal of Chromatography A, 1065 (2005) 121-128
One of the main forms of tomato pectin methylesterase (PME; EC 3.1.1.11) that is applicable to the food industry was isolated from fresh tomato fruit. The extraction of the PME isoenzymes involved washing the fresh tomato flesh with water in order to remove sugars and than solubilizing the enzymes with a diluted HCl solution at pH 1.6. The extract was then neutralized to pH 7.4 using buffer solution. After filtration, the solution was directly fractioned using Convective Interaction Media (CIM®) short monolithic disk column bearing sulfonyl (SO3) groups and using a linear gradient from 0 to 700 mM NaCl. The injection volume was 3 ml and the diameter of the column was 12 mm and length 3 mm. The isolated fractions were monitored for protein content and PME activity. The fraction with the targeted enzyme, which showed NaCl independent activity, was further purified and concentrated by ultrafiltration and finally purified by a second semi-preparative cation-exchange chromatography step using a CIM carboxymethyl (CM) disk monolithic column consisting of two disks and applying a step gradient. From 1 kg of fresh tomato fruits, 7.5 mg of purified PME with molecular mass estimated to be 26 000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) was obtained. A fraction with mixed PME and polygalacturonase activity was also obtained. Compared to the published procedures for the isolation and purification of PME from plant materials, this new procedure is much faster and more efficient. The potential application of CIM disk short monolithic columns in the analysis and semi-preparative extraction and isolation of the PME isoenzyme is presented.
G. A. Platonova, T. B. Tennikova
Journal of Chromatography A, 1065 (2005) 19–28(2005) 19–28
The technology for preparation of rigid macroporous polymers suggested in the late 1980s has become a powerful instrument for the development of a novel scientific and practical field. At present, monolithic stationary phases are widely used in the processes of bioseparation (chromatography), bioconversion (enzyme reactors) as well as in other processes based on interphase mass distribution (for example, solid phase peptide and oligonucleotide synthesis). Bioaffinity modes of suggested dynamic methods are very promising for their use in different analytical processes (immunological, ecological, medical and other types of analytical monitoring), preparative isolation of blood proteins such as myoglobin, hemoglobin, immunoglobulins, etc. and also recombinant products directly from cell supernatants or lysates. For the first time, it has been shown that bioaffinity pairing with participation of immobilized on carefully designed rigid supports is very fast and the whole process of affinity separation can be realized within second time scale. The principle of bioaffinity recognition is generaly at the construction of biological reactors (for example, enzyme reactors). Improved kinetics of biocatalized reactions is explained by a minimal influence on the surface of the used sorbent. Very perspective field is the use of discussed monoliths for solid phase chemical synthesis of fragments of biological macromolecules (peptides and oligonucleotides). Several examples of these applications will be presented and discussed.
G. A. Platonova, T. B. Tennikova
Journal of Chromatography A, 1065 (2005) 75–81(2005) 75–81
High-performance monolithic disk affinity chromatography was applied to the investigation of formation of complexes between (1) complementary polyriboadenylic and polyribouridylic acids, e.g. poly(A) and poly(U), respectively, (2) poly(A) and synthetic polycation poly(allylamine), pAA. Polyriboadenylic acid and poly(allylamine) were immobilized on macroporous disks (CIM disks). Quantitative parameters of affinity interactions between macromolecules were established using frontal analysis at different flow rates.
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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M. Benčina, K. Benčina, A. Štrancar, A. Podgornik
Journal of Chromatography A, 1065 (2005) 83–91(2005) 83–91
A deoxyribonuclease bioreactor was prepared by immobilization of deoxyribonuclease I through epoxy groups inherently present on poly (glycidyl methacrylate-co-ethylene dimethacrylate) monoliths. Columns with various levels of DNase activity were prepared varying immobilization temperature, pH, time and method. The apparent Michaelis–Menten constant, Kmapp, and turnover number, k3app, for immobilized DNase determined by on-line frontal analysis method were, respectively, 0.28 g of DNA l-1 and 16 dA260nm min-1 mg-1 of immobilized DNase. The highest activity of immobilized DNase was detected at 1 mM calcium ions concentration and mirrored properties of free enzyme; however, reaction temperature in the range from 25 to 37 °C has no significant effect on activity of immobilized DNase in contrary to free enzyme. The CIM DNase bioreactor was used for elimination of DNA contaminants in RNA samples prior to reverse transcription followed by PCR.
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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.
M. Bartolini, V. Cavrini, V. Andrisano
Journal of Chromatography A, 1065 (2005) 135-144
The aim of the present study was to optimize the preparation of an immobilized acetylcholinesterase (AChE)-based micro-immobilized enzyme reactor (IMER) for inhibition studies. For this purpose two polymeric monolithic disks (CIM, 3 mm × 12 mm i.d.) with different reactive groups (epoxy and ethylendiamino) and a packed silica column (3 mm × 5 mm i.d.; Glutaraldehyde-P, 40 μm) were selected as solid chromatographic supports. All these reactors were characterized in terms of rate of immobilization, stability, conditioning time for HPLC analyses, optimum mobile phase and peak shape, aspecific interactions and costs. Advantages and disadvantages were defined for each system. Immobilization through Schiff base linkage gave more stable reactors without any significant change in the enzyme behaviour; monolithic matrices showed very short conditioning time and fast recovery of the enzymatic activity that could represent very important features in high throughput analysis and satisfactory reproducibility of immobilization yield. Unpacked silica material allowed off-line low costs studies for the optimization of the immobilization step.
2004
A. Podgornik, J. Jančar, M. Merhar, S. Kozamernik, D. Glover, K. Čuček, M. Barut, A. Štrancar
J. Biochem. Biophys. Methods 60 (2004) 179–189
Monoliths represent a special class of chromatographic supports. In contrast to other stationary phases, they consist of a single piece of highly porous material through which a sample is mainly transported by convection. As a consequence, monoliths enable fast separations and exhibit flow-unaffected properties, which make them attractive for purification of macromolecules like proteins or DNA. In this work, methacrylate-based monolithic columns with the bed volume up to 8000 ml are characterized. They perform high-resolution separations of several hundreds of grams of proteins per hour by utilizing liter per minute flow rates. They are incompressible under these operating conditions and resistant to strong alkaline conditions.
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.
H. Podgornik, A. Podgornik
Journal of Chromatography B, 799 (2004) 343–347
Different chromatographic methods including chromatofocusing are used for separation of manganese peroxidase (MnP) isoforms and their isolation from the fungal growth medium. We tested strong anion exchange methacrylate based monolithic columns as a stationary phase for fast separation of MnP’s. Sodium acetate buffers of two different pH values (6 and 4) were used for formation of reproducible pH gradient. The entire cycle, involving analysis and column regeneration, was completed in 3 min. Use of pH gradient showed better MnP isoform separation comparing to the salt gradient, while application of combined pH–salt gradient, resulted in further improvement.