Miloš Barut

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.

Use of compact, porous units with immobilized ligands with high molecular masses in affinity chromatography and enzymatic conversion of substrates with high and low molecular masses

Different ligands with high molecular masses are immobilized on compact, porous separation units and used for affinity chromatography. In subsequent experiments different enzymes are immobilized and used for converting substrates with low and high molecular masses. Disk or tube with immobilized concanavalin A (ConA) are used as model systems for lectin affinity chromatography. The enzyme glucose oxidase is used as a standard protein to test the ConA units. Subsequently glycoproteins from plasma membranes of rat liver are separated, using units with immobilized ConA. The enzyme dipeptidyl peptidase i.v., which is used as a model protein in the experiments, is enriched about 40-fold in a single step, with a yield of over 90%. The results are only slightly better than those obtained with ConA when it is immobilized on bulk supports. The important improvement lies in the reduction of separation time to only 1 h. Experiments concerning the isolation of monoclonal antibodies against clotting factor VIII (FVIII) are carried out on disks, combining anion-exchange chromatography and protein A affinity chromatography as a model for multidimensional chromatography. Both IgG (bound to the protein A disk) and accompanying proteins (bound to the anion-exchange disk) from mouse ascites fluid are retarded and eluted separately. With the immobilized enzymes invertase and glucose oxidase (GOX) the corresponding substrates with low molecular masses, saccharose and glucose, are converted. It is shown that the amount of immobilized enzyme and the concentration of the substrate are responsible for the extent of the conversion, whereas the flow-rates used in the experiments have no effect at all. The influence of immobilization chemistry was investigated with GOX. Indirect immobilization with ConA as spacer proved to be the best alternative. With trypsin, immobilized on a disk, substrates with high molecular masses are digested in flow-through. For optimal digestion the proteins have to be denatured in the buffer for sodium dodecyl sulfate-polyacrlyamide gel electrophoresis prior to application. In contrast to the conversion of substrates with low molecular masses, flow-rates play an important part in conversion of substrates with high molecular masses. With lower flow-rates a higher degree of digestion is achieved.

Isocratic separations on thin glycidyl methacrylate–ethylenedimethacrylate monoliths

In this work, the isocratic separation of oligonucleotides in the ion-exchange mode on thin glycidylmethacrylate–ethylenedimethacrylate (GMA–EDMA) monoliths in the form of commercially available CIM (Convective Interaction Media) disks is presented. It was found that isocratic separation occurs even on monoliths with a thickness of only 0.75 mm. Peak broadening of the components retained on the monolith is proportional to the retention time, which in turn is proportional to the thickness of the monolith. Peak height is inversely proportional to the retention time. From these results it can be concluded that the mechanism of the separation on such monoliths is similar to that in HPLC columns filled with conventional porous particles. The height equivalent to a theoretical plate of GMA–EDMA monoliths is calculated to be 18.0 μm. The capacity factor k′ depends, exponentially, on the salt concentration. The Z factor calculated from fitted equations increases linearly with the oligonucleotide’s length. It was also found that the difference between peak retention volume slightly increases with the flow-rate when the experiments are performed in the range from 0.5 to 7 ml/min. From the similarities between the isocratic separations on conventional columns and on thin GMA–EDMA monoliths it is reasonable to believe that separation based on a multiple adsorption/desorption process also occurs in thin monoliths.

Application of short monolithic columns for improved detection of viruses.

Monolithic chromatography media represent a novel generation of stationary phases introduced in the last 10-15 years providing a chromatography matrix with enhanced mass transfer and hydrodynamic properties. These features allow for an efficient and fast separation of especially large biomolecules like e.g., DNA and viruses. In this study, the enrichment of virus RNA on short monolithic columns prior to molecular detection of viruses is described. Measles and mumps viruses were chosen as model viruses. The results show that it is possible to bind viral RNA on monoliths and concentrate viral nucleic acids from a fairly dilute sample. Consequently, a potential application of short monolithic columns is the concentration of virus RNA to improve the sensitivity and selectivity of viral detection with the possibility of isolating viral RNA from cell-free biological fluids.

Methacrylate‐based short monolithic columns: Enabling tools for rapid and efficient analyses of biomolecules and nanoparticles

This review describes the novel chromatography stationary phase--a porous monolithic methacrylate-based polymer--in terms of the design of the columns and some of the features that make these columns attractive for the purification of large biomolecules. We first start with a brief summary of the characteristics of these large molecules (more precisely large proteins like immunoglobulins G and M, plasmid deoxyribonucleic acid (DNA), and viral particles), and a list of some of the problems that were encountered during the development of efficient purification processes. We then briefly describe the structure of the methacrylate-based monolith and emphasize the features which make them more than suitable for dealing with large entities. The highly efficient structure on a small scale can be transferred to a large scale without the need of making column modifications, and the various approaches of how this is accomplished are briefly presented in this paper. This is followed by presenting some of the examples from the bioprocess development schemes, where the implementation of the methacrylate-based monolithic columns has resulted in a very efficient and productive process. Following this, we move back to the analytical scale and demonstrate the efficiency of the monolithic column--where the mass transfer between the stationary and mobile phase is greatly enhanced--for the in-process and final control of the new therapeutics. The combination of an efficient structure and the appropriate hardware results in separations of proteins with residence time less than 0.1 s.

Application of very short monolithic columns for separation of low and high molecular mass substances

ABSTRACT 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.

Application of monoliths for downstream processing of clotting factor IX

In this paper, the application of monolithic columns for downstream processing of different clotting factor IX concentrates is shown. Determination of basic chromatographic conditions as well as investigations on the regeneration of disk- and tube-shaped monolithic columns using human serum albumin as a model protein, were performed. Separation of factor IX and vitronectin, a possible impurity in commercial factor IX concentrates was accomplished using disk-shaped monolithic columns. These same applications were also carried out with identical results on up-scaled tube-shaped monolithic columns. Since these media allow very fast separations, this method can be successfully applied not only to an in-process control of the purification of factor IX but also to other biopolymers from human plasma. Besides, the same application on the up-scaled tube-shaped monolithic column was successfully carried out.

Application of semi-industrial monolithic columns for downstream processing of clotting factor IX

It has been shown in a previous study that monolithic columns can be used for downstream processing of different concentrates of clotting factor IX [K. Branović et al., J. Chromatogr. A 903 (2000) 21]. This paper demonstrates that such supports are useful tools also at an early stage of the purification process of factor IX from human plasma. Starting with the eluate after solid-phase extraction with DEAE-Sephadex, the use of monolithic columns has allowed much better purification than that achieved with conventional anion-exchange supports. The period of time required for separation is also much reduced. In up-scaling experiments, separations are carried out with 8, 80 and 500 ml columns. A volume of 1830 ml of DEAE-Sephadex eluate, containing a total of 27.6 g of protein and 48500 IU of factor IX is applied to the 500 ml monolithic column. This corresponds to a separation on a pilot scale. The results of this separation after up-scaling are comparable to those obtained with the 8 ml column on a laboratory scale.

Depletion of high-abundance proteins from human plasma using a combination of an affinity and pseudo-affinity column.

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These high-abundance proteins prevent the detection of low-abundance proteins which are potential markers for various diseases. The depletion of HSA and IgG is therefore essential for further proteome analysis. In this paper we describe the optimization of conditions for selective depletion of HSA and IgG using affinity and pseudo-affinity chromatography. A BIA Separations CIM (convective interaction media) Protein G disk was applied for the removal of IgG and the Mimetic Blue SA A6XL stationary phase for the removal of HSA. The binding and the elution buffer for CIM Protein G disk were chosen on the basis of the peak shape. The dynamic binding capacity was determined. It was shown to be dependent on the buffer system used and independent of the flow rate and of the concentration of IgG. Beside the binding capacity for the IgG standard, the binding capacity was also determined for IgG in human plasma. The Mimetic Blue SA A6XL column was characterized using human plasma. The selectivity of the depletion was dependent on the amount of human plasma that was loaded on the column. After the conditions on both supports had been optimized, the Mimetic Blue SA A6XL stationary phase was combined with the CIM Protein G disk in order to simultaneously deplete samples of human plasma. A centrifuge spin column that enables the removal of IgG and HSA from 20 microL of human plasma was designed. The results of the depletion were examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis.

Rapid high-performance liquid chromatographic analysis of adenovirus type 5 particles with a prototype anion-exchange analytical monolith column

To support effective process development there is a requirement for rapid analytical methods that can identify and quantitate adenoviral particles throughout the manufacturing process, from cellular lysate through to purified adenovirus. An anion-exchange high-performance liquid chromatography method for the analysis of adenovirus type 5 (Ad5) particles has been developed using a novel quaternary amine monolithic column (Bio-Monolith QA, Agilent). The developed method separates intact Ad5 from contaminating proteins and DNA, thus allowing analysis of non-purified samples during process development. Regeneration conditions were incorporated to extend the functional life of the column. Once developed, the method was qualified according to performance criteria of repeatability, intermediate precision and linearity. The linear working range of analysis was established between 7.5 x 10(8) to at least 2.4 x 10(10) viral particles (3 x 10(10) to 9.6 x 10(11) viral particles/mL), with a correlation coefficient of 0.9992. Relative standard deviations (RSDs) for intra- and inter-day repeatability and precision for retention time and peak area were less than 1 and 2.5%, respectively.