Publications
2008
I. V. Kalashnikova, N. D. Ivanova, T. B. Tennikova
Russian Journal of Applied Chemistry, 2008, Vol. 81, No. 5, pp. 867-873
A simple virus-cell complementary model system can be obtained using polymer-analogous reactions of the epoxy groups of glycidyl methacrylate-ethylene glycol dimethacrylate monolithic macroporous polymeric support and of the carboxy groups of styrene-methyl methacrylate polymeric nanospheres. The effect of thus designed microenvironment on the affinity binding parameters of virus-mimicking nanoparticles with the functionalized sorbent surface is studied by high-performance monolithic disk affinity chromatography.
S. Likić, G. Rusak, M. Krajačić
Journal of Chromatography A, 1189 (2008) 451–455
High-performance liquid chromatography was developed for further separation of double-stranded (ds) RNAs obtained by CF-11 cellulose chromatography from plants infected with satellite associated cucumber mosaic virus. Fractions separated by monolithic polymer column, especially applicable for nucleic acid analyses, were identified electrophoretically and confirmed with a polymerase chain reaction test. Once standardized, the method has revealed clear evidence of satellite presence without precipitation and electrophoresis. According to demonstrated sensitivity, its application in the preliminary diagnostics of field samples is also predictable. Principally, it can be used as a powerful preparative approach resulting in highly pure satellite dsRNA for further analyses.
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.
P. Kramberger, M. Peterka, J. Boben, M. Ravnikar, A. Štrancar
Journal of Chromatography A, 1144 (2007), pg. 143–149
Drawbacks of conventional virus purification methods have led to the development of new, mostly chromatography-based methods. Short monolithic columns are stationary phases intended for purification of large molecules. In this work efficient chromatographic purification of tomato mosaic virus (ToMV) from plant material is described. Based on short monolithic column, the purification process was shortened from 5 days to 2 hours. High viral purity was achieved and recovery of chromatographic step was up to 90%. In addition, these columns enabled preliminary quantification of the virus in just a few minutes, much faster than other quantification methods (e.g. enzyme-linked immunosorbent assay or real-time polymerase chain reaction) which take 1–2 days. These results demonstrate the potential of short monolith column technology for purification and analysis of different viruses.
J. Boben, P. Kramberger, N. Petrovič, K. Cankar, M. Peterka, A. Štrancar, M. Ravnikar
European Journal of Plant Pathology (2007) 118:59-71
A quantitative RT real-time PCR method was developed for the detection and quantification of Tomato mosaic virus (ToMV) in irrigation waters. These have rarely been monitored for the presence of plant pathogenic viruses, mostly due to the lack of efficient and sensitive detection methods. The newly developed method presented here offers a novel approach in monitoring the health status of environmental waters. ToMV was reliably detected at as low as 12 viral particles per real-time PCR reaction, which corresponds to the initial concentration of approximately 4.2 × 10-10 mg (6,300 viral particles) of ToMV per ml of sample. The sensitivity of the method was further improved by including the Convective Interaction Media® (CIM) monolithic chromatographic columns for quick and efficient concentration of original water samples. Seven out of nine water sources from different locations in Slovenia tested positive for ToMV, after concentrating the sample. Four samples tested ToMV-positive without the concentrating procedure. The presence and integrity of infective ToMV particles in the original sample, as well as in the chromatographic fraction, was confirmed using different methods from test plants, DAS ELISA to electron microscopy and real-time PCR. In this study, we propose a unique and simple diagnostic scheme for rapid, efficient, and sensitive monitoring of irrigation waters that could also be adopted for other plant, human or animal viruses.
M. Peterka, D. Glover, P. Kramberger, M. Banjac, A. Podgornik, M. Barut, A. Štrancar
BioProcessing Journal, March/April 2005
The last 30 years have seen rapid and dramatic developments in recombinant DNA technology and the related biological sciences. In 1972, Paul Berg's group used restriction enzymes to cut DNA in half and then used ligases to stick the pieces of the DNA back together. By doing this, they produced the first recombinant DNA. Within a year, the first genetically engineered bacterium existed. A little more than ten years later, recombinant human insulin was approved for diabetic patients and became the first recombinant healthcare product. Before the end of the 1980s, the first gene therapy trial had occurred. Today, a large number of recombinant proteins are used as marketed drugs and even more are in clinical trials targeting a wide range of diseases.
2005
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. 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
P. Kramberger, N. Petrovič, A. Štrancar, M. Ravnikar
Journal of Virological Methods 120 (2004) 51-57120 (2004) 51-57
A new chromatographic medium, Convective Interaction Media® (CIM) disk monolithic columns, was applied to plant virus concentration. The ability of the columns to concentrate highly diluted plant viruses was tested on a model plant virus, rod-shaped tomato mosaic virus (ToMV). Enzyme-linked immunosorbent assay (ELISA) was used for the quantitative analysis. The virus was concentrated using a strong anion exchanger, CIM quaternary amine (QA) disk monolithic column. A high salt concentration was used to elute the concentrated virus from the columns. It has been demonstrated that ToMV, which had been diluted considerably below the sensitivity of ELISA, was concentrated by several orders of magnitude in the one-step procedure. Concentrated virus preparations could be used directly for ELISA testing. In comparison with methods described for concentrating plant viruses from irrigation water, the above procedure may provide a much faster and more efficient way to concentrate highly diluted plant viruses. The procedure could be applied to the testing of other highly diluted plant viruses, and to concentrating viruses for antiserum production.
2003
P. Kramberger, D. Glover, A. Štrancar
American Biotechnology Laboratory, 2003, 21(13), 27-8.
Research in molecular and cell biology has shown that macromolecules such as pDNA and virus vectors, together called nanoparticles, have the potential to assist in the prevention and treatment of some human diseases. The most important step in their production is the downstream processing (isolation and cleaning). Precipitation, ultrafiltration, and LC techniques are the most widely used for these purposes, but only LC can purify the product so that it is recognized as safe for therapeutic use. Apart from reduced yield, downstream processing can cause minor or even major modifications in the structure of the biomolecule. Usually these modifications do not affect the activity of the product, but may change its antigenicity. Minimizing these changes to maintain product safety is the main objective in the downstream processing of nanoparticles. For the efficient isolation of labile biomolecules, liquid chromatographic supports should provide fast and efficient separation in order to decrease biomolecule degradation; have high, preferably flow-unaffected capacity and resolution; and exhibit low backpressure. They should be stable, even if harsh conditions are applied during sanitation (e.g., 1 M NaOH), and should be easy to handle and operate. CIM® (Convection Interaction Media) monolithic chromatographic columns (BIA Separations, Ljubljana, Slovenia) meet all of these requirements. This article will discuss the columns and their use on human models and plant viruses and pDNA.