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.
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.
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; 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.
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.
R. Giovannini, R. Freitag, T. B. Tennikova
Anal. Chem. 1998, 70, 3348-3354
Membrane adsorbers are well established in protein chromatography. The present paper investigated for the first time the behavior of polynucleotides on these stationary phases, taking a 7.2-kb predominantly supercoiled plasmid as example. Gradient and isocratic elution was studied. In contrast to protein high-performance membrane chromatography (HPMC), isocratic elution is possible in DNA chromatography. In the case of gradient elution, much higher salt concentrations can be used in the starting buffer. Under optimized conditions, both approaches led to a splitting of the single plasmid peak into three maximums, which corresponded to the threealbeit isolated bands in the agarose gel. Presumably the three fractions were supercoiled, nicked, and open circular plasmid DNA. Linearization of the plasmid lowered the adsorption energy, and the linearized plasmid eluted earlier than the nonlinearized one. The HPMC experiments were compared to similar ones performed using a conventional packed-bed anion-exchange column (BioScale Q2, 7 × 52 mm, 10-μm porous particles) and a novel monolithic-type anion-exchange column (UNO Q1, 7 × 35 mm). The results and characteristic differences observed in these experiments were interpreted in the light of the newly developed theory of HPMC.