Publications
2009
I. Gutiérrez-Aguirre, M. Banjac, A. Steyer, M. Poljšak-Prijatelj, M. Peterka, A. Štrancar, M. Ravnikar
Journal of Chromatography A, 1216 (2009) 2700–2704
Rotaviruses are the leading cause of diarrhoea in infants around the globe and, under certain conditions they can be present in drinking water sources and systems. Ingestion of 10–100 viral particles is enough to cause disease, emphasizing the need for sensitive diagnostic methods. In this study we have optimized the concentration of rotavirus particles using methacrylate monolithic chromatographic supports. Different surface chemistries and mobile phases were tested. A strong anion exchanger and phosphate buffer (pH 7) resulted in the highest recoveries after elution of the bound virus with 1 M NaCl. Using this approach, rotavirus particles spiked in 1 l volumes of tap or river water were efficiently concentrated. The developed concentration method in combination with a real time quantitative polymerase chain reaction assay detected rotavirus concentrations as low as 100 rotavirus particles/ml.
2008
M. Ćurković Perica, I. Šola, L. Urbas, F. Smrekar, M. Krajačić
Journal of Chromatography A 1216 (2009) 2712-2716
A procedure based on Sartorius BIA Separations CIM DEAE anion-exchange chromatography was developed to separate double-stranded (ds) RNA of hypovirus infecting phytopathogenic fungus Cryphonectria parasitica. Using a linear gradient of 25 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0 as a binding buffer, and 25 mM MOPS, 1.5 M NaCl, 0.1 mM EDTA, 15% isopropanol (v/v), pH 7.0 as an elution buffer, hypoviral dsRNA was additionally purified from nucleic acid species present in preparations partially purified by standard CF-11 cellulose chromatography. Moreover, crude phenol/chloroform extracts of the fungal tissue were also applied to monolithic supports and CIM DEAE chromatograms revealed clear evidence for hypoviral presence without CF-11 chromatography, nucleic acid precipitation, and electrophoresis.
J. Ivancic-Jelecki, M. Brgles, M. Šantak, D. Forčić
Journal of Chromatography A 1216 (2009) 2717-2724
Human plasma is an important medical substance and a raw material for production of various therapeutics. During blood sampling, storage and processing, genomic DNA is released into plasma from nucleated blood cells that are damaged in the course of the procedure. In order to determine the concentration of contaminating DNA in plasma, we developed a method for DNA isolation by using anion-exchange chromatography on a Sartorius BIA Separations CIM (convective interaction media) diethylaminoethyl column. DNA was quantified by SYBR Green based real-time polymerase chain reaction. The concentration of cell-free, non-apoptotic DNA in plasma ranged between 0.06 and 22.5 ng/ml. As substantial volumes of plasma or whole blood are administered directly into the vascular system, a recipient is exposed to high amounts of cell-free DNA, several orders of magnitude higher than the amount found in other biologicals.
F. Smrekar, M. Ciringer, M. Peterka, A. Podgornik, A. Štrancar
Journal of Chromatography B, (2007)
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.
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
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.
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.
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
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.
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.