Ajdovščina, Slovenia
Ajdovščina, Slovenia

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Racki N.,Slovenian National Institute of Biology | Kramberger P.,BIA Separations | Steyer A.,University of Ljubljana | Gaspersic J.,BIA Separations | And 3 more authors.
Journal of Chromatography A | Year: 2015

Enteric viruses are commonly present in environmental waters and represent the major cause of waterborne infections and outbreaks. Since traditional wastewater treatments fail to remove enteric viruses in the water purification process, they are released daily into environmental waters. Monolithic supports have enabled chromatography to enter the field of virology. They have been successfully used in virus purification and concentration. In this work quaternary amine (QA) methacrylate monoliths were exploited to remove enteric viruses from wastewater treatment plant effluent. Expectedly, chromatographic processing of such a complex medium was troublesome, even for monoliths, characterized by extremely large pore dimensions. This problem was solved by introducing a pre-step chromatography using hydroxyl (OH) methacrylate monoliths. This way, molecules, that would hinder virus binding to the anion-exchanger monolith, were removed. As a result, the OH pre-column reduced backpressure increase on the subsequent anion-exchanger column, and increased both QA column binding capacity and life time. Wastewater effluent samples were successfully purified from five waterborne enteric viruses (rotavirus, norovirus genogroup I and II, astrovirus, sapovirus), below the detection limit of RT-qPCR. The breakthrough of the rotavirus binding capacity was not reached for concentrations that significantly exceeded those expected in effluent waters. The obtained results confirm that methacrylate monoliths can be a valuable tool for simultaneous removal of different waterborne viruses from contaminated water sources. © 2015 The Authors.


Peljhan S.,BIA Separations | Vidic J.,BIA Separations | Krajnc N.L.,BIA Separations | Fonovic M.,Jozef Stefan Institute | Ropret P.,Smithsonian Institution
Microchemical Journal | Year: 2016

ELISA (Enzyme-linked ImmunoSorbent Assay), as one possible application of antibody-based methods, has proved to be a good candidate for the determination of proteins in works of art due to the low limit of detection, high sensitivity (in the nanogram range), specificity and micro invasiveness of required sampling. Despite the advantages of this method, interference by metal ions present in pigments, lipids, saccharides and in other components of paints, grounds, and varnishes may cause several problems for incontestable identification of proteins. To overcome the drawbacks of ELISA, a novel way to purify the extracted mixtures to predominantly protein fractions, by the DEAE-modified (weak anion exchange) CIM® (Convective Interaction Media) chromatographic monoliths was introduced. Utilisation of monolithic chromatographic supports greatly enhanced the ELISA detection of ovalbumin in the model paint samples (aged and non-aged), for at least one tenfold dilution of a sample. In several cases the purification enabled detection of the target protein, otherwise not possible with non-purified samples. For the first time in the field of Cultural Heritage, the combined approach of ELISA and CIM® monolith-supported extraction was successfully tested also for the detection of protein in real-case paint layers. © 2016 Elsevier B.V.


PubMed | Ruder Boskovic Institute, University of Zagreb, University Paris - Sud and BIA Separations
Type: Journal Article | Journal: Journal of separation science | Year: 2016

The serotype specificity of adenovirus ion-exchange chromatography has previously been studied using standard particle-based columns, and the hexon protein has been reported to determine retention time. In this study, we have submitted Adenovirus type 5 recombinants to anion-exchange chromatography using methacrylate monolithic supports. Our experiments with hexon-modified adenoviral vectors show more precisely that the retention time is affected by the substitution of amino acids in hypervariable region 5, which lies within the hexon DE1 loop. By exploring the recombinants modified in the fiber protein, we have proven the previously predicted chromatographic potential of this surface constituent. Modifications that preserve the net charge of the hexon protein, or those that cause only a small charge difference in the fiber protein, in addition to shortening the fiber shaft, did not change the chromatographic behavior of the adenovirus particles. However, modifications that include the deletion of just two negatively charged amino acids in the hexon protein, or the introduction of a heterologous fiber protein, derived from another serotype, revealed recognizable changes in anion-exchange chromatography. This could be useful in facilitating chromatography-approach purification by creating targeted capsid modifications, thereby shifting adenovirus particles away from particular interfering substances present in the crude lysate.


Bednar I.,University of Natural Resources and Life Sciences, Vienna | Bednar I.,Austrian Center of Industrial Biotechnology | Berger E.,Austrian Center of Industrial Biotechnology | Krajnc N.L.,BIA Separations | And 5 more authors.
Langmuir | Year: 2014

Seven porous chromatographic columns, termed monoliths, and seven nonporous sheets were produced from polymethacrylates. Their surfaces were activated by different densities of butyl and phenyl ligands. We determined the retention times of highly dilute molecular probes in monoliths and accessed contact angles of pure molecular probes of sheets. We calculated surface energies for both systems. We applied theories of Young, Dupré, and van Oss and compared the results of both types of experiments with respect to Lifshitz-van der Waals and Lewis acid and Lewis base contributions and find agreement but an additive constant. © 2014 American Chemical Society.


Bednar I.,Austrian Center of Industrial Biotechnology | Tscheliessnig R.,Austrian Center of Industrial Biotechnology | Berger E.,Austrian Center of Industrial Biotechnology | Podgornik A.,BIA Separations | And 2 more authors.
Journal of Chromatography A | Year: 2012

Hydrophobicity of hydrophobic interaction chromatography media is currently ranked according to retention of reference proteins. A new method, suitable for porous media, is presented here to determine the surface energy and its Lifshitz-van-der-Waals, Lewis acid and Lewis base contributions. The theory of van Oss has been adapted for data obtained by inverse liquid chromatography. Furthermore, this method is characterized by the independence of the determination of the phase ratio. The retention of probes with different molecular properties was used to calculate the surface energy and the Lifshitz-van-der-Waals as well as Lewis acid and Lewis base contributions to the surface energy. The media with polymethacrylate backbone had a higher surface energy (γ≈200 mJ/m2) and Lifshitz-van-der-Waals contribution (γLW≈140mJ/m2) than the agarose-based media (γ≈90-180mJ/m2 and γLW≈50-160mJ/m2). © 2011 Elsevier B.V.


Limonta M.,Center for Genetic Engineering and Biotechnology | Krajnc N.L.,BIA Separations | Vidic U.,BIA Separations | Zumalacarregui L.,Polytechnic University José Antonio Echeverría
Biochemical Engineering Journal | Year: 2013

The pIDKE2 plasmid is the main component of the CIGB's candidate vaccine against Hepatitis C virus (HVC), which is being used in HCV chronically-infected individuals during clinical trials phase 1 and 2. The designed downstream process of pIDKE2 plasmid produces up to 179. g/year. In order to conduct further improvements, modelling of the downstream process was performed. A methodology based on process analysis tools, such as experimental design and modelling was established to identify factors with the highest influence on production cost and the amount of annual plasmid. Taking into account that the pIDKE2 downstream process designed is in its initial stages of development, CIM technology was evaluated as a new manufacturing process on lab scale. Purity and recovery of CIM technology was better than porous particle matrix, thus SuperPro Designer was used in order to simulate the purification process. Cost efficiency optimization of the pIDKE2 downstream process was done with the simulation model. © 2013 Elsevier B.V.


Banjac M.,BIA Separations | Roethl E.,Green Hills | Gelhart F.,Green Hills | Kramberger P.,BIA Separations | And 5 more authors.
Vaccine | Year: 2014

We explored the possibilities for purification of various δNS1 live, replication deficient influenza viruses on ion exchange methacrylate monoliths. Influenza A δNS1-H1N1, δNS1-H3N2, δNS1-H5N1 and δNS1-influenza B viruses were propagated in Vero cells and concentrated by tangential flow filtration. All four virus strains adsorbed well to CIM QA and CIM DEAE anion exchangers, with CIM QA producing higher recoveries than CIM DEAE. δNS1-influenza A viruses adsorbed well also to CIM SO3 cation exchanger at the same pH, while δNS1-influenza B virus adsorption to CIM SO3 was not complete. Dynamic binding capacity (DBC) for CIM QA, DEAE and SO3 methacrylate monoliths for influenza A δNS1-H1N1 virus were 1.9E+10 TCID50/ml, 1.0E+10 TCID50/ml and 8.9E+08 TCID50/ml, respectively. Purification of δNS1 viruses on CIM QA was scaled up and reproducibility was confirmed. Yields of infectious virus on CIM QA were between 70.8±32.3% and 87±30.8%. Total protein removal varied from 93.3±0.4% to 98.6±0.2% and host cell DNA removal efficiency was ranging from 76.4% to 99.9% and strongly depended on pretreatment with deoxyribonuclease. © 2014 Elsevier Ltd.


Kramberger P.,BIA Separations | Urbas L.,BIA Separations | Strancar A.,BIA Separations
Human Vaccines and Immunotherapeutics | Year: 2015

Downstream processing of nanoplexes (viruses, virus-like particles, bacteriophages) is characterized by complexity of the starting material, number of purification methods to choose from, regulations that are setting the frame for the final product and analytical methods for upstream and downstream monitoring. This review gives an overview on the nanoplex downstream challenges and chromatography based analytical methods for efficient monitoring of the nanoplex production. © 2015 Taylor & Francis Group, LLC.


PubMed | BIA Separations
Type: | Journal: Virology journal | Year: 2012

The NanoSight LM10 with Nanoparticle tracking analysis (NTA) software was evaluated for the quantification of latex particles, adenovirus 5, and influenza virus. The inter-day variability was determined by measuring the same sample over several consecutive days and the methods accuracy was demonstrated by using known concentrations of the subject particles. NTA analysis was also used to quantify chromatographic fractions of adenovirus and influenza virus after purification on a CIM monolithic column. NTA results were compared and evaluated against hemagglutination (HA) and end point dilution assay, determining total and infection virus particle number, respectively. The results demonstrated that nanoparticle tracking analysis is a method for fast estimation of virus concentration in different samples. In addition, it can provide a better insight into the sample status, regarding the level of virus aggregation.

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