Publications
2008
C. Delattre, P. Michaud, M. A. Vijayalakshmi
Journal of Chromatography B, 861 (2008) 203–208
Fast production and purification of α-(1,4)-oligogalacturonides was investigated using a new enzymatic reactor composed of a monolithic matrix. Pectin lyase from Aspergillus japonicus (Sigma) was immobilized on CIM-disk epoxy monolith. Studies were performed on free pectin lyase and immobilized pectin lyase to compare the optimum temperature, optimum pH, and thermal stability. It was determined that optimum temperature for free pectin lyase and immobilized pectin lyase on monolithic support is 30 °C, and optimum pH is 5. Monolithic CIM-disk chromatography is one of the fastest liquid chromatographic method used for separation and purification of biomolecules due to high mass transfer rate. In this context, online one step production and purification of oligogalacturonides was investigated associating CIM-disk pectin lyase and CIM-disk DEAE. This efficient enzymatic bioreactor production of uronic oligosaccharides from polygalacturonic acid (PGA) constitutes an original fast process to generate bioactive oligouronides.
2007
R. Hahn, A. Tscheliessnig, P. Bauerhansl, A. Jungbauer
J. Biochem. Biophys. Methods 70 (2007) 87–94
Monolithic media have found widespread use as excellent tools for fast analytical separations of small molecules, proteins, pDNA and viruses. Polymethacrylate monoliths with large channels are attractive for capturing large molecules, like immunoglobulins, DNA, and viruses. For preparative purposes, these monoliths are operated in radial flow mode. Band spreading in monoliths is extremely low and mostly dominated by the contribution of extra column effects. The model used here had a single axial dispersion coefficient which lumps together extra column effects and the intrinsic band spreading of the monolithic material to characterize the adsorption of proteins and pDNA on polymethacrylate ion-exchange monoliths. Due to the fact that the performance of the monolith was unaffected by the velocity within the applied range, and due to highly favourable adsorption isotherms, a constant pattern model could be applied to predict preparative runs on radial flow units assuming axial flow for modelling.
R. Skudas, B. A. Grimes, E. Machtejevas, V. Kudirkaite, O. Kornysova, T. P. Hennessy, D. Lubda, K. K. Unger
Journal of Chromatography A, 1144 (2007) 72-84(2007) 72 - 84
In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of ∼25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18 < C8 < C4. The resolution of proteins and peptides by reversed-phase gradient liquid chromatography on n-octadecyl, n-octyl, and n-butyl bonded monolithic silica columns is optimized. The results obtained imply the use of acetonitrile concentration gradient up to 75% for n-octadecyl and n-octyl bonded monolithic silica columns, and the use of acetonitrile concentration gradient up to 85% for n-butyl bonded monolithic silica columns. With the respect to the gradient times and flow rates, the optimum conditions are the best with n-octyl and n-butyl bonded monolithic silica columns, where the range of optimum gradient times is up to ∼30 min and mobile phase flow rates in the range of 0.5–1 ml/min. Consequently, the best performance towards peak resolution is obtained with n-octyl bonded monolithic silica column with the respect to low concentration of organic phase gradient, fast separations and low solvent consumptions due to low flow rates.
J. Vidič, A. Podgornik, J. Jančar, V. Frankovič, B. Košir, N. Lendero, K. Čuček, M. Krajnc, A. Štrancar
Journal of Chromatography A, 1144 (2007) 63–71(2007) 63 – 71
Chemical and chromatographic stability of methacrylate-based monolithic columns bearing 3-N,N-diethylamino-2-hydroxypropyl (DEAE) and quarternary amine (QA) groups was studied. The leakage products from both monolithic columns were determined and the leakage of amines has been quantified in alkali solutions. Monolithic columns bearing QA functional groups being exposed to 1 M sodium hydroxide solution for up to 3 months caused reduction of ion-exchange groups for approximately 12%, while for DEAE monolithic columns was only around 3% in 1 year. In 0.1 M NaOH and 20% ethanol degradation was significantly lower. The main leaking compound from DEAE monolith was found to be 3-(diethylamino)-1,2-propanediol and 2,3-dihydroxypropyltrimethylammonium salt for QA monolith. During repeated 50 cleaning-in-place (CIP) cycles, no changes in chromatographic properties were detected.
B. A. Grimes, R. Skudas, K. K. Unger, D. Lubda
Journal of Chromatography A, 1144 (2007) 14-29(2007) 14-29
In this work, a parallel pore model (PPM) and a pore network model (PNM) are developed to provide a state-of-art method for the calculation of several characteristic pore structural parameters from inverse size-exclusion chromatography (ISEC) experiments. The proposed PPM and PNM could be applicable to both monoliths and columns packed with porous particles. The PPM and PNM proposed in this work are able to predict the existence of the second inflection point in the experimental exclusion curve that has been observed for monolithic materials by accounting for volume partitioning of the polymer standards in the macropores of the column. The appearance and prominence of the second inflection point in the exclusion curve is determined to depend strongly on the void fraction of the macropores (flow-through pores), (b) the nominal diameter of the macropores, and (c) the radius of gyration of the largest polymer standard employed in the determination of the experimental ISEC exclusion curve. The conditions that dictate the appearance and prominence of the second inflection point in the exclusion curve are presented. The proposed models are applied to experimentally measured ISEC exclusion curves of six silica monoliths having different macropore and mesopore diameters. The PPM and PNM proposed in this work are able to determine the void fractions of the macropores and silica skeleton, the pore connectivity of the mesopores, as well as the pore number distribution (PND) and pore volume distribution (PVD) of the mesopores. The results indicate that the mesoporous structure of all materials studied is well connected as evidenced by the similarities between the PVDs calculated with the PPM and the PNM, and by the high pore connectivity values obtained from the PNM. Due to the fact that the proposed models can predict the existence of the second inflection point in the exclusion curves, the proposed models could be more applicable than other models for ISEC characterization of chromatographic columns with small diameter macropores (interstitial pores) and/or large macropore (interstitial pore) void fractions. It should be noted that the PNM can always be applied without the use of the PPM, since the PPM is an idealization that considers an infinitely connected porous medium and for materials having a low (<6) pore connectivity the PPM would force the PVD to a lower average diameter and larger distribution width as opposed to properly accounting for the network effects present in the real porous medium.
I. Junkar, T. Koloini, P. Krajnc, D. Nemec, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1144 (2007) 48-54(2007) 48-54
Today, monoliths are well-accepted chromatographic stationary phases due to several advantageous properties in comparison with conventional chromatographic supports. A number of different types of monoliths have already been described, among them recently a poly(high internal phase emulsion) (PolyHIPE) type of chromatographic monoliths. Due to their particular structure, we investigated the possibility of implementing different mathematical models to predict pressure drop on PolyHIPE monoliths. It was found that the experimental results of pressure drop on PolyHIPE monoliths can best be described by employing the representative unit cell (RUC) model, which was originally derived for the prediction of pressure drop on catalytic foams. Models intended for the description of particulate beds and silica monoliths were not as accurate. The results of this study indicate that the PolyHIPE structure under given experimental condition is, from a hydrodynamic point of view, to some extent similar to foam structures, though any extrapolation of these results may not provide useful predictions of pressure versus flow relations and further experiments are required.
K. Isobe, Y. Kawakami
Journal of Chromatography A, 1144 (2007) 85-89(2007) 85-89
A convection interaction media (trade name CIM, Sartorius BIA Separation, Ljubljana, Slovenia) isobutyl monolithic disc was prepared by incubating a CIM epoxy monolithic disc with isobutylamine, and it was then applied to the purification of secondary alcohol dehydrogenase (S-ADH) and primary alcohol oxidase (P-AOD). Both enzymes were adsorbed on this column and eluted with high purity. Thus, S-ADH was purified to an electrophoretically homogeneous state by four column chromatographies using CIM DEAE-8 and CIM C4-8 tube monolithic columns, blue-Sepharose column and CIM isobutyl disc monolithic column. P-AOD was also purified to an electrophoretically homogeneous state by three column chromatographies of CIM DEAE-8 tube, CIM C4-8 tube and CIM isobutyl disc columns.
S. Laschober, M. Sulyok, E. Rosenberg
Journal of Chromatography A, 1144 (2007) 55-62(2007) 55-62
The present work aims at the optimisation of the synthesis of methyl-silsesquioxane monolithic capillary columns using a sol–gel based protocol. The influence of reaction conditions such as temperature, reaction mixture composition and catalyst concentration has been examined. The morphology of the products was studied by scanning electron microscopy and nitrogen adsorption. Monolithic capillary columns were obtained with a skeleton-like structure with open pores. Pore diameters vary from 0.8 to 15 μm, diameters of the xerogel network vary from 0.4 to 12 μm, respectively. Specific surface areas up to 334 m2/g have been observed, however, many materials did not possess areas above few m2/g which represents the limit of detection of the nitrogen porosimetry measurements. Excellent adhesion to the capillary wall was observed in all cases, and drying was possible at ambient conditions without the formation of cracks.
I.Vovk, B. Simonovska
Journal of Chromatography B, 849 (2007) 337-343
The most abundant isoforms of tomato pectin methylesterase (PME; EC 3.1.1.11; Mr 26 kDa), polygalacturonase (PG; EC 3.2.1.15; PG1 with Mr 82 kDa) and a basic protein with Mr 42 kDa and unknown function were isolated from fresh tomato fruit by a fast chromatographic procedure on a Convective Interaction Media (CIM®) short monolithic disk column bearing carboxymethyl (CM) groups. The extraction of the targeted enzymes with 1.2 M NaCl solution was followed by precipitation with ammonium sulfate at 60% of saturation, solubilisation of the pellet in 0.5 M NaCl and fractionation using a linear gradient from 0 to 700 mM NaCl. Among six fractions five had PME activity and four had PG activity, while one fraction containing a pure protein with Mr 42 kDa with neither of these activities. Two concentrated fractions, one with PG and one with PME were further purified. A linear gradient from 0 to 500 mM NaCl with 20% CH3CN in the mobile phase was used for the PG fraction and two CM disks and a linear gradient from 0 to 200 mM NaCl were used for the PME fraction as a greater capacity was necessary in this case. From 4 kg of fresh tomato flesh we obtained 22 mg of purified PME, 1.8 mg of purified, active PG1, 13.5 mg of additional basic protein and a fraction with PG2 contaminated by a PME isoform. Carboxymethyl CIM disk short monolithic columns are convenient for semi-preparative and analytical work with tomato fruit pectolytic enzymes.
I. Vovk, B. Simonovska
Journal of Chromatography A, 1144 (2007) 90-96(2007) 90-96
An improved cation-exchange chromatographic procedure on Convective Interaction Media (CIM, Sartorius BIA Separations, Ljubljana, Slovenia) short monolithic methacrylate disk columns was used for the isolation of salt-independent pectin methylesterase (PME; EC 3.1.1.11) isoform and endo-polygalacturonase PG1 (PG, EC 3.2.1.15) from ripe tomato fruit extract after studying the chromatographic conditions including type of disk, binding buffer, pH, eluent composition and different gradients. Between 10 and 20 μg of proteins gave reliable chromatograms. Both carboxymethyl (CM) and sulfonyl (SO3) disks were equally suitable for the fractionation of tomato extract using the new gradient, but only CM disk was appropriate for further purification of the PME and PG fractions, and provided fast and sharp separation of proteins. The isolation of pure PG1 could be achieved only by addition of 20% of acetonitrile to the mobile phase. About 200 μg of proteins were loaded at one chromatographic run at the fractionation and purification. Determination of the molecular weights of the separated proteins showed that dimer of salt-independent PME isoform was formed in concentrated solutions of the enzyme but dissociated upon dilution of the solution. From 6 kg of fresh tomato flesh, 28 mg of purified salt-independent PME, 12.5 mg of purified and active PG1 and 4 mg of PG2 fraction contaminated with salt-dependent PME isoform were obtained by means of semi-preparative chromatography on CIM disks.
E. Machtejevas, S. Andrecht, D. Lubda, K. K. Unger
Journal of Chromatography A, 1144 (2007) 97-101
The following particulate and monolithic silica columns were implemented in a fully automated and flexible multidimensional LC/MS system with integrated sample clean-up, to perform the analysis of endogeneous peptides from filtered urine and plasma samples: restricted access sulphonic acid strong cation-exchanger (RAM-SCX) for sample clean-up, RP 18 Chromolith guard columns as trap columns and 100 μm I.D. monolithic RP 18 fused silica capillary columns as last LC dimension. The results show sufficient overall system reproducibility and repeatability. Implementation of monolithic silica columns added an additional flexibility with respect to flow rate variation and adjustment due to the low column back pressures. Also, monolithic columns showed a lower clogging rate in long-term usage for biological samples as compared to particulate columns. The applied system set-up was tested to be useful for the routine peptide screening in search of disease biomarkers.
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.
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.
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.
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
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.