2007
F. SMREKAR, M. CIRINGER, M. PETERKA, A. PODGORNIK, A. STRANCAR. Purification and concentration of bacteriophage T4 using monolithic chromatographic supports. J Chromatogr B Analyt Technol Biomed Life Sci., 2007, Jun 7.
Phages are gaining importance due to their wide usage. In this work strong anion exchange monolithic chromatographic column was used for single step phage purification. Most of the proteins and DNA were removed and recovery of approximately 70% of infective virus was reproducibly achieved. 30 ml of phage sample was purified in around 10 min.
K. BENČINA, M. BENČINA, A. PODGORNIK, A. ŠTRANCAR. Influence of the methacrylate monolith structure on genomic DNA mechanical degradation, enzymes activity and clogging. J Chromatogr A, 2007,1160, 176-83.
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 143nm pore radius mechanically damaged the DNA and was clogged at flow rates above 0.5mlmin(-1) (26cmh(-1)). For monoliths with a pore radius of 634nm and 2900nm, no mechanical degradation of DNA was observed up to 5mlmin(-1)(265cmh(-1)) above which the HPLC itself became the main source of damage. A decrease of a permeability appeared at flow rate 1.8mlmin(-1)(95cmh(-1)) and 2.3mlmin(-1) (122cmh(-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 634nm 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.
J. CHAMPAGNE, C. DELATTRE, C. SHANTHI, B. SATHEESH, L. DUVERNEUIL,M. A. VIJAYALAKSHMI. Pseudoaffinity Chromatography Using a Convective Interaction Media® Disk Monolithic Column. Chromatographia, 2007, 65, 639-648.
Monolithic convective interaction media-disk (CIM-disk) chromatography is one of the fastest liquid chromatographic methods for the separation and purification of biomolecules due to its high mass transfer rate. In this way, all separated molecules are transported by convection into the pores of the matrix, resulting in a very fast separation due to the low mass transfer resistance of the CIM-disk. Due to the advantages of monolith technique, in recent years, CIM-disk affinity chromatography has been developed and investigated for purification of peptides, restriction enzymes, antibodies, etc. In this review, applications of monolithic affinity chromatography are discussed. The purification of restriction enzymes, polyclonal and monoclonal antibodies using a new monolithic CIM-disk system with immobilized histidine affinity chromatography are presented.
R. HAHN, A. TSCHELIESSNIG, P. BAUERHANSL, A. JUNGBAUER. Dispersion effects in preparative polymethacrylate monoliths operated in radial-flow columns. J. Biochem. Biophys. Methods, 2007, 70, 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.
T. ČERK PETRIČ, P. BRNE, B. GABOR, L. GOVEDNIK, M. BARUT, A. ŠTRANCAR, L. ZUPANČIČ KRALJ. Anion-exchange chromatography using short monolithic columns as a complementary technique for human serum albumin depletion prior to human plasma proteome analysis. J Pharm Biomed Anal., 2007, 43, 243-9.
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.