Biomedical subjects

Ales Podgornik

Publications and source records attributed to Ales Podgornik.

At least 19 recordsLinked to original sources

PubMed2006

Sequential injection affinity chromatography utilizing an albumin immobilized monolithic column to study drug-protein interactions.

In this study, sequential injection affinity chromatography was used for drug-protein interactions studies. The analytical system used consisted of a sequential injection analysis (SIA) manifold directly connected with convective interaction media (CIM) monolithic epoxy disks modified by ligand-immobilization of protein. A non-steroidal, anti-inflammatory drug, naproxen (NAP) and bovine serum albumin (BSA) were selected as model drug and protein, respectively. The SIA system was used for sampling, introduction and propulsion of drug towards to the monolithic column. Association equilibrium constants, binding capacity at various temperatures and thermodynamic parameters (free energy DeltaG, enthalpy DeltaH) of the binding reaction of naproxen are calculated by using frontal analysis mathematics. The variation of incubation time and its effect in on-line binding mode was also studied. The results indicated that naproxen had an association equilibrium constant of 2.90 x 10(6)M(-1) at pH 7.4 and 39 degrees C for a single binding site. The associated change in enthalpy (DeltaH) was -27.36 kcal mol(-1) and the change in entropy (DeltaS) was -73 cal mol(-1)K(-1) for a single type of binding sites. The location of the binding region was examined by competitive binding experiments using a biphosphonate drug, alendronate (ALD), as a competitor agent. It was found that the two drugs occupy the same class of binding sites on BSA. All measurements were performed with fluorescence (lambda(ext)=230 nm, lambda(em)=350 nm) and spectrophotometric detection (lambda=280 nm).

Animals↗

PubMed2006

Chemical and chromatographic stability of methacrylate-based monolithic columns.

Chemical and chromatographic stability of methacrylate-based monolithic columns bearing 3-N,N-diethylamino-2-hydroxypropyl (DEAE) and quarternary amine (QA) groups was studied. The leakage products from both monolithic columns were determined and the leakage of amines has been quantified in alkali solutions. Monolithic columns bearing QA functional groups being exposed to 1M sodium hydroxide solution for up to 3 months caused reduction of ion-exchange groups for approximately 12%, while for DEAE monolithic columns was only around 3% in 1 year. In 0.1M NaOH and 20% ethanol degradation was significantly lower. The main leaking compound from DEAE monolith was found to be 3-(diethylamino)-1,2-propanediol and 2,3-dihydroxypropyltrimethylammonium salt for QA monolith. During repeated 50 cleaning-in-place (CIP) cycles, no changes in chromatographic properties were detected.

Chromatography, Ion Exchange↗

PubMed2006

Incorporation of a monolithic column into sequential injection system for drug-protein binding studies.

A sequential injection analysis (SIA) manifold was incorporated with a monolithic strong anion-exchanger disk for on-line drug-protein interaction studies. The antibiotic ciprofloxacin (CF) was selected as a model drug compound. The separation principle was based on the strong retention of bovine serum albumin (BSA) on the monolithic strong anion-exchanger and the liberation/release of the free form of the drug. Elution of the retained BSA was easily achieved by delivering a different mobile phase via the SIA manifold. The type of functional group of the monolithic support, the breakthrough volume and the injected volumes of CF and BSA were studied and optimized. The influence of the variation of incubation time was studied in on-line binding assays. Scatchard plot was employed to obtain the number of binding sites and the equilibrium binding constants. For the off-line study of the CF-BSA binding, two binding classes were determined with constants of (3.16+/-0.21)x10(6)M(-1) and (1.27+/-0.48)x10(4)M(-1) and 6.1+/-1.3 and 17.8+/-3.9 binding sites per class, respectively. In non-equilibrium binding experiments the binding rate constant was k(1)=785 M(-1)min(-1). All measurements were monitored with fluorescence (lambda(ext)=300 nm, lambda(em)=460 nm) and spectrophotometric detection (lambda=280 nm). To evaluate the accuracy of the developed method the obtained results were compared versus ultrafiltration experiments and were found in good agreement.

Anion Exchange Resins↗

PubMed2005

Detection of processed genetically modified food using CIM monolithic columns for DNA isolation.

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 pretreated 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.

Chromatography, Ion Exchange↗

PubMed2005

Immobilization of deoxyribonuclease via epoxy groups of methacrylate monoliths. Use of deoxyribonuclease bioreactor in reverse transcription-polymerase chain reaction.

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, Km(app), 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 degrees 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.

Base Sequence↗

PubMed2005

Preparation and characterisation of poly(high internal phase emulsion) methacrylate monoliths and their application as separation media.

Poly(glycidyl methacrylate-co-ethyleneglycol dimethacrylate) monolithic supports were prepared by radical polymerisation of the continuous phase of water in oil high internal phase emulsions. Morphology of monolithic materials was studied by scanning electron microscopy and mercury intrusion porosimetry. The ratio of phase volume and the degree of crosslinking influenced the void size and pore size distribution of resulting polymers. Void sizes between 1 and 10 microm were observed and average pore sizes around 100 nm. Polymers with 60, 75, 80 and 90% pore volume were prepared and even samples with highest pore volume showed good mechanical stability. They were modified to bear weak-anion exchange groups and tested on the separation of standard protein mixture containing myoglobin, conalbumine and trypsin inhibitor. Good separation was obtained in a very short time similar to the separation obtained by commercial methacrylate monoliths. However, higher dispersion was observed. Bovine serum albumin dynamic binding capacity for monolith with 90% porosity was close to 9 mg/ml.

Microscopy, Electron, Scanning↗

PubMed2005

Pressure drop in CIM disk monolithic columns.

Pressure drop analysis in commercial CIM disk monolithic columns is presented. Experimental measurements of pressure drop are compared to hydrodynamic models usually employed for prediction of pressure drop in packed beds, e.g. free surface model and capillary model applying hydraulic radius concept. However, the comparison between pressure drop in monolith and adequate packed bed give unexpected results. Pressure drop in a CIM disk monolithic column is approximately 50% lower than in an adequate packed bed of spheres having the same hydraulic radius as CIM disk monolith; meaning they both have the same porosity and the same specific surface area. This phenomenon seems to be a consequence of the monolithic porous structure which is quite different in terms of the pore size distribution and parallel pore nonuniformity compared to the one in conventional packed beds. The number of self-similar levels for the CIM monoliths was estimated to be between 1.03 and 2.75.

Chromatography↗

PubMed2005

Simple method for determining the amount of ion-exchange groups on chromatographic supports.

The objective of this study was to develop a fast, simple, non-destructive, non-toxic and low-priced method for determining the amount of ionic groups on resins, since the conventional titration method fails to give proper results on methacrylate monoliths. After the column had been pre-saturated with a high concentration buffer solution, a low concentration buffer solution of the same pH value was pumped through the column. Measuring pH and absorbance, the profiles with a shape of typical break-through curve were obtained. It was shown that the time of the pH transient, which appeared under such conditions, could be used as a measure of the total ionic capacity of ion-exchange monolithic columns. The effect of the column length, linear velocity and varying concentrations of buffer solutions on the time of the pH transient was examined. The method was shown to be suitable for determining the amount of ionic groups on both anion and cation monolithic columns. In addition, it could also be applied to particle bed columns. The time of the pH transient and the protein dynamic binding capacity were also compared and it was concluded that for a given monolith the protein capacity can be derived from the data obtained by the new method.

Chromatography, Ion Exchange↗

PubMed2005

Effect of the glass surface modification on the strength of methacrylate monolith attachment.

The influence of glass surface modification in order to determine strength of the monolith attachment was studied. Modification consists of pre-treatment of the glass with chemicals or boiling in deionized water, silanization and drying has been investigated on different types of glass. Amount of silane groups was determined by measurement of the contact angle between the glass surface and water drop. The highest values were found for soda-lime glass. Strength of the monolith attachment was established by pumping ethanol through the monolithic capillaries and measuring the pressure drop at which monolith was dislodged. Surprisingly, it was found that the critical part of the glass surface modification procedure is glass pre-treatment. Good results were obtained with glass boiled in water for 2.5 h or more.

Glass↗

PubMed2005

Application of monoliths for plasmid DNA purification development and transfer to production.

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 2001 fermentation aliquots pilot scale purification runs were performed in order to prove scale-up and to predict further up-scaling to 8 1 tube monolithic columns. The analytical results obtained from these runs confirmed suitability for pharmaceutical applications.

Chromatography, Ion Exchange↗

PubMed2005

Convective interaction media short monolithic columns: enabling chromatographic supports for the separation and purification of large biomolecules.

New therapeutics that are being developed rely more and more on large and complex biomacromolecules like proteins, DNA, and viral particles. Manufacturing processes are being redesigned and optimized both upstream and downstream to cope with the ever-increasing demand for the above target molecules. In downstream processing, LC still represents the most powerful technique for achieving high yield and high purities of these molecules. In most cases, however, the separation technology relies on conventional particle-based technology, which has been optimized for the purification of smaller molecules. New technologies are, therefore, needed in order to push the downstream processing ahead and into the direction that will provide robust, productive, and easy to implement methods for the production of novel therapeutics. New technologies include the renaissance of membranes, various improvements of existing technologies, but also the introduction of a novel concept--the continuous bed or monolithic stationary phases. Among different introduced products, Convective Interaction Media short monolithic columns (SMC) that are based on methacrylate monoliths exhibit some interesting features that make them attractive for these tasks. SMC can be initially used for fast method development on the laboratory scale and subsequently efficiently transferred to preparative and even more importantly to industrial scale. A brief historical overview of methacrylate monoliths is presented, followed by a short presentation of theoretical considerations that had led to the development of SMC. The design of these columns, as well as their scale-up to large units, together with the methods for transferring gradient separations from one scale to another are addressed. Noninvasive methods that have been developed for the physical characterization of various batches of SMC, which fulfill the regulatory requirements for cGMP production, are discussed. The applications of SMC for the separation and purification of large biomolecules, which demonstrate the full potential of this novel technology for an efficient downstream processing of biomolecules, are also presented.

Biopolymers↗

PubMed2005

Convective Interaction Media (CIM)--short layer monolithic chromatographic stationary phases.

Modern downstream processing requires fast and highly effective methods to obtain large quantities of highly pure substances. Commonly applied method for this purpose is chromatography. However, its main drawback is its throughput since purification, especially of large molecules, requires long process time. To overcome this problem several new stationary phases were introduced, among which short layer monoliths show superior properties for many applications. The purpose of this review is to give an overview about short methacrylate monolithic columns commercialised under the trademark Convective Interaction Media (CIM). Their unique properties are described from different perspectives, explaining reasons for their application on various areas. Approaches to prepare large volume methacrylate monolithic column are discussed and optimal solutions are given. Different examples of CIM monolithic column implementation are summarised in the last part of the article to give the reader an idea about their advantages.

Algorithms↗

PubMed2004

Separation of manganese peroxidase isoenzymes on strong anion-exchange monolithic column using pH-salt gradient.

Different chromatographic methods including chromatofocusing are used for separation of manganese peroxidase (MnP) isoforms and their isolation from the fungal growth medium. We tested strong anion exchange methacrylate based monolithic columns as a stationary phase for fast separation of MnP's. Sodium acetate buffers of two different pH values (6 and 4) were used for formation of reproducible pH gradient. The entire cycle, involving analysis and column regeneration, was completed in 3 min. Use of pH gradient showed better MnP isoform separation comparing to the salt gradient, while application of combined pH-salt gradient, resulted in further improvement.

Anion Exchange Resins↗

PubMed2004

Characterization of methacrylate monoliths for purification of DNA molecules.

The suitability of methacrylate based anion exchange monolithic supports for the separation and purification of plasmid and genomic DNA has been explored. The effect of the size of the channels, ionic strength of the solution, and ligand density on the dynamic binding capacity has been investigated. The dynamic binding capacity was found to be flow independent, at least up to a linear velocity of 700 cm h(-1), and exceeded 9 mg mL(-1) for all types of DNA. The recovery depends on the pH value of the mobile phase and its ionic strength as well as on the density of the active groups. Under optimal conditions recoveries exceeding 80% were obtained even for genomic DNA. Finally, the suitability of this approach is demonstrated by purification of a real-life sample.

Aspergillus niger↗

PubMed2004

Enzyme immobilization on epoxy- and 1,1'-carbonyldiimidazole-activated methacrylate-based monoliths.

Monolithic Convective Interaction Media (CIM) have been activated with epoxide and imidazole carbamate functionalities and used as supports for covalent immobilization of protein A, deoxyribonuclease I, and trypsin. The efficiency of immobilization for these proteins was determined from the amount of bound IgG, degradation of DNA, and hydrolysis of Nalpha-benzoyl-L-arginine ethyl ester, respectively. The respective biological activities of trypsin and the binding capacity of protein A immobilized via imidazole carbamate groups were 11.45 and 2.25 times higher than those obtained for epoxide matrix while they were practically equal for deoxyribonuclease I. The kinetics of immobilization was studied in detail for trypsin under dynamic conditions and revealed that the enzyme immobilized via imidazole carbamate groups already reached its highest activity in 5 min. In contrast, a much longer time was required for immobilization via epoxy groups.

Animals↗

PubMed2003

Characterization of CIM monoliths as enzyme reactors.

The immobilization of the enzymes citrate lyase, malate dehydrogenase, isocitrate dehydrogenase and lactate dehydrogenase to CIM monolithic supports was performed. The long-term stability, reproducibility, and linear response range of the immobilized enzyme reactors were investigated along with the determination of the kinetic behavior of the enzymes immobilized on the CIM monoliths. The Michaelis-Menten constant K(m) and the turnover number k(3) of the immobilized enzymes were found to be flow-unaffected. Furthermore, the K(m) values of the soluble and immobilized enzyme were found to be comparable. Both facts indicate the absence of a diffusional limitation in immobilized CIM enzyme reactors.

Enzymes, Immobilized↗

PubMed2003

Temperature influence on the dynamic binding capacity of a monolithic ion-exchange column.

This work investigates the influence of temperature on the binding capacity of bovine serum albumin (BSA), soybean trypsin inhibitor and L-glutamic acid to a CIM (DEAE) weak anion-exchange disk monolithic column. The binding capacity was determined experimentally under dynamic conditions using frontal analysis. The effect on the dynamic binding capacity of dimers present in the BSA solution has been evaluated and a closed-loop frontal analysis was used to determine the equilibrium binding capacities. The binding capacity for both BSA and soybean trypsin inhibitor increased with increasing temperature. In the case of L-glutamic acid, an increase in the binding capacity was observed with temperature up to 20 degrees C. A further increase in temperature caused a decrease of the dynamic binding capacity.

Anion Exchange Resins↗

PubMed2002

Short monolithic columns as stationary phases for biochromatography.

Monolithic supports represent a novel type of stationary phases for liquid and gas chromatography, for capillary electrochromatography, and as supports for bioconversion and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, monolithic supports are cast as continuous homogeneous phases. They represent an approach that provides high rates of mass transfer at lower pressure drops as well as high efficiencies even at elevated flow rates. Therefore, much faster separations are possible and the productivity of chromatographic processes can be increased by at least one order of magnitude as compared to traditional chromatographic columns packed with porous particles. Besides the speed, the nature of the pores allows easy access even in the case of large molecules, which make monolithic supports a method of choice for the separation of nanoparticles like pDNA and viruses. Finally, for the optimal purification of larger biomolecules, the chromatographic column needs to be short. This enhances the speed of the separation process and reduces backpressure, unspecific binding, product degradation and minor changes in the structure of the biomolecule, without sacrificing resolution. Short Monolithic Columns (SMC) were engineered to combine both features and have the potential of becoming the method of choice for the purification of larger biomolecules and nanopartides on the semi-preparative scale.

Biomedical Engineering↗