Publications
2007
M. Benčina, J. Babič, A. Podgornik
Journal of Chromatography A, 1144 (2007) 135–142
In gene therapy and DNA vaccination, RNA removal from DNA preparations is vital and is typically achieved by the addition of ribonuclease into the sample. Removal of ribonuclease from DNA samples requires an additional purification step. An alternative is the implementation of immobilized ribonuclease. In our work, ribonuclease was covalently coupled onto the surface of methacrylate monoliths via epoxy or imidazole carbamate groups. Various immobilization conditions were tested by changing immobilization pH. Ribonuclease immobilized on the monolith via imidazole carbamate groups at pH 9 was found to be six times more active than the ribonuclease immobilized on the monolith via epoxy groups. Under optimal immobilization conditions the Michaelis–Menten constant, Km, for cytidine-2,3-cyclic monophosphate, and turnover number, k3 were 0.52 mM and 4.6 s-1, respectively, and mirrored properties of free enzyme. Enzyme reactor was found to efficiently eliminate RNA contaminants from DNA samples. It was active for several weeks of operation and processed 300 column volumes of sample. Required residence time to eliminate RNA was estimated to be around 0.5 min enabling flow rates above 1 column volume per min.
R. Nicoli, N. Gaud, C. Stella, S. Rudaz, J.-L. Veuthey
Journal of Pharmaceutical and Biomedical Analysis 48 (2008) 398–407
The preparation and characterization of three trypsin-based monolithic immobilized enzyme reactors (IMERs) developed to perform rapid on-line protein digestion and peptide mass fingerprinting (PMF) are described. Trypsin (EC 3.4.21.4) was covalently immobilized on epoxy, carboxy imidazole (CDI) and ethylenediamine (EDA) Convective Interaction Media® (CIM) monolithic disks. The amount of immobilized enzyme, determined by spectrophotometric measurements at 280 nm, was comprised between 0.9 and 1.5 mg per disk. Apparent kinetic parameters K*m and V*max, as well as apparent immobilized trypsin BAEE-units, were estimated in flow-through conditions using N-α-benzoyl-l-arginine ethyl ester (BAEE) as a low molecular mass substrate. The on-line digestion of five proteins (cytochrome c, myoglobin, α1-acid glycoprotein, ovalbumin and albumin) was evaluated by inserting the IMERs into a liquid chromatography system coupled to an electrospray ionization ion-trap mass spectrometer (LC-ESI–MS/MS) through a switching valve. Results were compared to the in-solution digestion in terms of obtained scores, number of matched queries and sequence coverages. The most efficient IMER was obtained by immobilizing trypsin on a CIM® EDA disk previously derivatized with glutaraldehyde, as a spacer moiety. The proteins were recognized by the database with satisfactory sequence coverage using a digestion time of only 5 min. The repeatability of the digestion (R.S.D. of 5.4% on consecutive injections of myoglobin 12 μM) and the long-term stability of this IMER were satisfactory since no loss of activity was observed after 250 injections.
M. Bartolini, V. Cavrini, V. Andrisano
Journal of Chromatography A, 1144 (2007) 102–110
The aim of the present study was the application of a human AChE-CIM-IMER (enzyme reactor containing acetylcholinesterase immobilized on a monolithic disk) for the rapid evaluation of the thermodynamic and kinetic constants, and the mechanism of action of new selected inhibitors. For this application, human recombinant AChE was covalently immobilized onto an ethylenediamine (EDA) monolithic Convective Interaction Media (CIM) disk and on-line studies were performed by inserting this IMER into a HPLC system. Short analysis time, absence of backpressure, low nonspecific matrix interactions and immediate recovery of enzyme activity were the best characteristics of this AChE-CIM-IMER. Mechanisms of action of selected reversible inhibitors (tacrine, donepezil, edrophonium, ambenonium) were evaluated by means of Lineweaver–Burk plot analysis. Analyses were performed on-line by injecting increasing concentrations of the tested inhibitor and substrate and by monitoring the product peak area. AChE-CIM-IMER kinetic parameters (Kmapp and vmaxapp ) were derived as well as inhibitory constants (Kiapp of selected compounds. Moreover, noteworthy results were obtained in the application of the AChE-CIM-IMER to the characterization of the carbamoylation and decarbamoylation steps in pseudo-irreversible binding of carbamate derivatives (physostigmine and rivastigmine). AChE-CIM-IMER appeared to be a valid tool to determine simultaneously the kinetic constants in a reliable and fast mode. The obtained values were found in agreement with those obtained with the classical methods with the free enzyme. Furthermore, after inactivation by carbamates, activity could be fully recovered and the AChE-CIM-IMER could be reused for further studies. Results showed that the AChE-CIM-IMER is a valid tool not only for automated fast screening in the first phase of the drug discovery process but also for the finest characterization of the mode of action of new hit compounds with increased accuracy and reproducibility and with saving of time and materials.
2005
M. Barut, A. Podgornik, P. Brne, A. Štrancar
J. Sep. Sci. 2005, 28, 1876-1892
New therapeutics that are being developed rely more and more on large and complex biomacromolecules like proteins, DNA, and viral particles. Manufacturing processes are being redesigned and optimized both upstream and downstream to cope with the ever-increasing demand for the above target molecules. In downstream processing, LC still represents the most powerful technique for achieving high yield and high purities of these molecules. In most cases, however, the separation technology relies on conventional particle-based technology, which has been optimized for the purification of smaller molecules. New technologies are, therefore, needed in order to push the downstream processing ahead and into the direction that will provide robust, productive, and easy to implement methods for the production of novel therapeutics. New technologies include the renaissance of membranes, various improvements of existing technologies, but also the introduction of a novel concept – the continuous bed or monolithic stationary phases. Among different introduced products, Convective Interaction Media short monolithic columns (SMC) that are based on methacrylate monoliths exhibit some interesting features that make them attractive for these tasks. SMC can be initially used for fast method development on the laboratory scale and subsequently efficiently transferred to preparative and even more importantly to industrial scale. A brief historical overview of methacrylate monoliths is presented, followed by a short presentation of theoretical considerations that had led to the development of SMC. The design of these columns, as well as their scale-up to large units, together with the methods for transferring gradient separations from one scale to another are addressed. Noninvasive methods that have been developed for the physical characterization of various batches of SMC, which fulfill the regulatory requirements for cGMP production, are discussed. The applications of SMC for the separation and purification of large biomolecules, which demonstrate the full potential of this novel technology for an efficient downstream processing of biomolecules, are also presented.
A. Podgornik, A. Štrancar
Biotechnology Annual Review, 11 (2005) 281-333
Modern downstream processing requires fast and highly effective methods to obtain large quantities of highly pure substances. Commonly applied method for this purpose is chromatography. However, its main drawback is its throughput since purification, especially of large molecules, requires long process time. To overcome this problem several new stationary phases were introduced, among which short layer monoliths show superior properties for many applications. The purpose of this review is to give an overview about short methacrylate monolithic columns commercialised under the trademark Convective Interaction Media® (CIM). Their unique properties are described from different perspectives, explaining reasons for their application on various areas. Approaches to prepare large volume methacrylate monolithic column are discussed and optimal solutions are given. Different examples of CIM monolithic column implementation are summarised in the last part of the article to give the reader an idea about their advantages.
A. Jungbauer
Journal of Chromatography A, 1065 (2005) 3–12
Bioseparation processes are dominated by chromatographic steps. Even primary recovery is sometimes accomplished by chromatographic separation, using a fluidized bed instead of a fixed bed. In this review, the action principles, features of chromatography media regarding physical and chemical properties will be described. An attempt will be made to establish categories of different media. Characteristics for bioseparation are the large pores and particle sizes. To achieve sufficient capacity for ultralarge molecules, such as plasmids or nanoparticles, such as viruses monoliths are the media of choice. In these media, the mass transport is accomplished by convection, and thus, the low diffusivity can be overcome. Common to all modern chromatography media is the fast operation. There are examples where a residence time of less then 3 min, is sufficient to reach the full potential of the adsorbent.
P. Krajnc, N. Leber, D. Štefanec, S. Kontrec, A. Podgornik
Journal of Chromatography A, 1065 (2005) 69-73(2005) 69 - 73
Poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) monolithic supports were prepared by radical polymerisation of the continuous phase of water in oil high internal phase emulsions. Morphology of monolithic materials was studied by scanning electron microscopy and mercury intrusion porosimetry. The ratio of phase volume and the degree of crosslinking influenced the void size and pore size distribution of resulting polymers. Void sizes between 1 and 10 μm were observed and average pore sizes around 100 nm. Polymers with 60, 75, 80 and 90% pore volume were prepared and even samples with highest pore volume showed good mechanical stability. They were modified to bear weak-anion exchange groups and tested on the separation of standard protein mixture containing myoglobin, conalbumine and trypsin inhibitor. Good separation was obtained in a very short time similar to the separation obtained by commercial methacrylate monoliths. However, higher dispersion was observed. Bovine serum albumin dynamic binding capacity for monolith with 90% porosity was close to 9 mg/ml.
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.
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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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.
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.
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.