Publications
2005
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.
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.
2002
A. Podgornik, M. Barut, S. Jakša, J. Jančar, A. Štrancar
Journal of Liquid Chromatography & Related Technologies Vol. 25, No. 20, pp. 3097–3114, 2002
Convective Interaction Media® (CIM) disk monolithic columns are specific among the chromatographic columns because of their monolithic structure and extremely short column length. In this work, HETP values and Z factors for different groups of molecules—proteins, DNA, oligonucleotides, peptides, and organic acids on strong anion exchange CIM disk monolithic columns were determined. Results are discussed in terms of the molecule structures and applied to develop different approaches for successful separation of abovementioned group of molecules on these types of columns.
R. Hahn, M. Panzer, E. Hansen, J. Mollerup, A. Jungbauer
Separation Science and Technology, 37(7), 1545–1565 (2002)
The mass transfer properties of polyglycidylmethacrylate–ethylenedimethacrylate monolithic ion-exchangers (convective interaction media disks) were evaluated. As a reference material, the particulate ion-exchanger Source 30 was selected. The model proteins lysozyme, bovine serum albumin, and IgG were loaded at different concentrations and velocities. The mass transfer zones obtained with the monoliths were affected by neither the linear flow velocity nor the protein concentration in the mobile phase. The reduced height equivalent to one theoretical plate (HETP) of monoliths were independent of the reduced velocity. This was not the case for the particulate material.
A. Štrancar, A. Podgornik, M. Barut, D. Glover
BIOforum International 3/2002
In adsorptive chromatographic modes, the slope of the capacity factor k' (defined as the molar ratio of the separated compound in the stationary phase and the mobile phase) plot versus composition of the mobile phase is very steep. Up to a certain composition of the mobile phase, k' is so high that the protein is bound to the stationary phase and does not move along the column. Reaching a defined point, a small change of the mobile phase composition causes a rapid decrease in k' to a value near zero. At this point, the protein dissolves in the mobile phase and passes through the column practically without any retention. In other words, the protein remains adsorbed at the top of the column until the eluting power of the mobile phase reaches the point at which a small change in the composition of the mobile phase causes the movement of the protein without any retention. One can also speak about selective elution of the compound. As a result of this process, even very short columns can provide very good separations and recovery, while longer columns might cause problems due to unspecific binding, product degradation and minor changes in the structure of the protein which increase with the length of the column. On the other hand, short-beds are very difficult to pack with particles and form channels which eliminate the resolution power of the column. Monolithic supports offer an ideal solution to avoid most of these problems.
K. Pflegerl, A. Podgornik, E. Berger, A. Jungbauer
Biotechnology and Bioengineering 79 (2002) 733-740
Screening of peptide ligands for affinity chromatography usually involves incubation with the target protein in a batch system. In an additional step, peptides with fast binding kinetics have to be selected in respect to satisfactory performance under flow conditions on a support ensuring optimal three-dimensional presentation of the peptide. We have developed a rapid screening system based on peptide synthesis and screening on CIM® disks. The disk size was minimized to fit into microplates usually applied for solid-phase extraction. In combination with a vacuum manifold, semi-automated peptide synthesis and screening for binding to a target protein under simulated chromatography conditions are possible. Various analytical methods can be applied for parallel and automated determination of the quantity, integrity, or activity of the target protein in the flow through or bound to the affinity support. This system also allows parallel screening for suitable chromatographic conditions like running buffer, washing, and elution conditions. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 79: 733–740, 2002.
T. Tennikova, A. Štrancar
LabPlus international February - March 2002, Volume 16
Monolithic supports are a novel generation of stationary phases that can be used for liquid and gas chromatography, capillary electrochromatography, bioconversions, as well as supports for solid phase synthesis. In contrast to individual particles packed into chromatographic columns, monolithic supports are cast as continuous homogeneous phases. They provide high rates of mass transfer at lower pressure drops and enable much faster separations. In addition to the speed, the nature of the pores allows easy permeability for large molecules. Monolithic supports are thus the method of choice for the separation of proteins, oligonucleotides, and nanoparticles such as pDNA and viruses. In this article we review the application of the monlithic columns to bioaffinity chromatography.