Publications
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.
2005
M. Barut, A. Podgornik, P. Brne, A. Štrancar
J. Sep. Sci. 2005, 28, 1876-1892
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.
A. Podgornik, A. Štrancar
Biotechnology Annual Review, 11 (2005) 281-333
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.
A. Jungbauer
Journal of Chromatography A, 1065 (2005) 3–12
Bioseparation processes are dominated by chromatographic steps. Even primary recovery is sometimes accomplished by chromatographic separation, using a fluidized bed instead of a fixed bed. In this review, the action principles, features of chromatography media regarding physical and chemical properties will be described. An attempt will be made to establish categories of different media. Characteristics for bioseparation are the large pores and particle sizes. To achieve sufficient capacity for ultralarge molecules, such as plasmids or nanoparticles, such as viruses monoliths are the media of choice. In these media, the mass transport is accomplished by convection, and thus, the low diffusivity can be overcome. Common to all modern chromatography media is the fast operation. There are examples where a residence time of less then 3 min, is sufficient to reach the full potential of the adsorbent.
P. Krajnc, N. Leber, D. Štefanec, S. Kontrec, A. Podgornik
Journal of Chromatography A, 1065 (2005) 69-73(2005) 69 - 73
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 μm 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.
N. Lendero, J. Vidič, P. Brne, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1065 (2005) 29-38(2005) 29 - 38
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.
I. Mihelič, D. Nemec, A. Podgornik, T. Koloini
Journal of Chromatography A, 1065 (2005) 59-67(2005) 59 - 67
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.
T. B. Tennikova, J. Reusch
Journal of Chromatography A, 1065 (2005) 13-17(2005) 13 - 17
The history of the development of short monolithic beds is described.
J. Vidić, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1065 (2005) 51-58(2005) 51-58
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.
S. Yamamoto, A. Kita
Journal of Chromatography A, 1065 (2005) 45-50(2005) 45-50
Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.
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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2004
A. Podgornik, J. Jančar, M. Merhar, S. Kozamernik, D. Glover, K. Čuček, M. Barut, A. Štrancar
J. Biochem. Biophys. Methods 60 (2004) 179–189
Monoliths represent a special class of chromatographic supports. In contrast to other stationary phases, they consist of a single piece of highly porous material through which a sample is mainly transported by convection. As a consequence, monoliths enable fast separations and exhibit flow-unaffected properties, which make them attractive for purification of macromolecules like proteins or DNA. In this work, methacrylate-based monolithic columns with the bed volume up to 8000 ml are characterized. They perform high-resolution separations of several hundreds of grams of proteins per hour by utilizing liter per minute flow rates. They are incompressible under these operating conditions and resistant to strong alkaline conditions.
D. G. Glover, M. Barut, A. Podgornik, M. Peterka, A. Štrancar
BioProcess International, Oct 2004, 58-63
The sequencing of the human genome and the rise of proteomics have increased the numbers of potential therapeutic targets. Biotechnology companies need to increase productivity, decrease discovery and production costs, and use technologies that easily transfer across departments if they wish to remain competitive. The most important tools are those for separation (purification) of target substance(s). They should be easy to use and offer an identical performance and purification profile no matter where they are implemented — in discovery, production, or quality assurance (QA).
CIM Convective Interaction Media short monolithic columns are just such a unifying technology. Produced in shapes and sizes from microliter to liter scale, they represent an evolutionary approach to meeting biochromatographic separation requirements in research and product development. Able to withstand 1 M NaOH with no loss of capacity or resolution, these easily scalable columns have been optimized for analysis and cGMP production of complex biomolecules ranging from oligonucleotides and plasmid DNA (pDNA) to proteins and viruses.
P. N. Nesterenko, M. A. Rybalko
Mendeleev Commun. 2004
The continuous flow gradient and its effect on chromatographic parameters were investigated for the separations of inorganic anions on a monolithic porous disk with bonded hydroxyproline residues.
2003
E. G. Vlakh, G. A. Platonova, G. P. Vlasov, C. Kasper, A. Tappe, G. Kretzmer, T. B. Tennikova
Journal of Chromatography A, 992 (2003) 109–119
The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-l-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.
I. Mihelič, T. Koloini, A. Podgornik
Journal of Applied Polymer Science, Vol. 87, 2326-2334 (2003)
Monolithic stationary phases are becoming increasingly important in the field of liquid chromatography. Methacrylate-based monoliths are produced via free-radical bulk polymerization. The preparation of large-volume monoliths is a major problem because the intensive heat released during polymerization causes distortion of the porous monolithic structure. This work presents experimental measurements of temperature distributions during polymerization in moulds of different sizes and at various experimental conditions. A mathematical model for the prediction of temporal and spatial temperature distribution during the polymerization of methacrylate-based monolithic columns is introduced. The polymerization is described by an unsteady-state heat conduction equation with the generation of heat related to the general kinetics of polymerization. Predictions from the mathematical model are in good agreement with the experimental measurements at different experimental conditions. A method for construction of large-volume monolithic columns is presented and an attempt is made to adopt the developed mathematical model in annular geometry.
P. Milavec Žmak, H. Podgornik, J. Jančar, A. Podgornik, A. Štrancar
Journal of Chromatography A, 1006 (2003) 195–205
Convective Interaction Media (CIM) columns are monolithic columns optimized for the separation of macromolecules. Some of them operate in the axial mode while others operate in the radial mode depending on the column size. In this work we tested the approach suggested by Yamamoto [Biotechnol. Bioeng., 48 (1995) 444] for transfer of gradient methods between columns of different size. A simplified equation for transfer was derived together with a criterion for its application. Separation was evaluated for a standard protein mixture and peroxidase enzymes present in fermentation broth. Salt and pH gradients were applied. Similar resolutions were obtained for each sample on all columns which demonstrates that the proposed approach can be successfully used for method scale-up on this type of column.
M. Merhar, A. Podgornik, M. Barut, M. Žigon, A. Štrancar
J. Sep. Sci. 2003, 26, 322–330
Methacrylate-based monoliths are formed during radical copolymerization as a consequence of the precipitation of polymeric chains from the reaction mixture, which consists of monomers, initiator, and the porogenic solvents. The effect of various methacrylate monomers on the porous structure of the monolith was investigated. Although the chemical structure of the monomers significantly affects the size of the pores and the porosity, the mechanism of pore formation in the case of the precipitation during polymerization is preserved. The porous structure was further correlated with the specific surface area, pressure drop, and dynamic binding capacity of the monoliths studied.
A. Podgornik, M. Barut, A. Štrancar
Encyclopedia of Chromatography DOI: 10.1081/E-ECHR 120016288, 2003
Chromatographic columns are typically several centimeters in length, resulting in a high number of column plates, and, consequently, such columns have high efficiency. These properties allow even very similar molecules to be separated. This is especially true for smaller molecules, where the separation is based on selective migration. For large molecules, a different separation mechanism is usually required. Large molecules normally interact with the matrix at several binding sites. Consequently, their adsorption isotherms are very steep, almost rectangular. For such molecules, there exists only a very narrow mobile phase range within which they interact with the active moieties on the stationary phase, but are not irreversibly retained. To elute them from the matrix, a change of the mobile phase composition is required. Therefore the separation is based upon the selective elution and requires the use of gradient chromatographic methods. For this type of separation, the column length is less important and the efficient separations can be achieved even with extremely short columns.
P. Kramberger, D. Glover, A. Štrancar
American Biotechnology Laboratory, 2003, 27-28
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 MNaOH), 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 application note will discuss the columns and their use on human models and plant viruses and pDNA.