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2003

Gene therapy which is becoming more and more important in human health care requires the purification of high molecular mass compounds, so called nanoparticles (e. g. viruses and plasmids). The method of choice to ensure proper purity would be chromatography.

Most of the chromatographic supports available on the market at the moment can not follow the requests for such work due to low binding capacity for large molecules, limitation with regards to the time of the separation process and requests for CIP (cleaning in place) and SIP (sanitation in place).

Monolithic supports represent a new generation of chromatographic supports. In contrast to conventional particle supports, where the void volume between individual porous particles is unavoidable, these supports consist of a single monolith highly interconnected with larger and smaller open flow-through channels. Due to the structure, molecules to be separated are transported to the active sites on the stationary phase by convection, resulting in very short separation times. This is especially true for large molecules.

In this work we will present the use of monolithic supports for the separation of different nanoparticles on analytical and preparative scales. It will be shown that monolithic supports can overcome the limitations of particle-based supports for the analytics and isolation of big molecules and represent a major step towards the safe and efficient purification or production of nanoparticles.

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Traces of DNA in RNA samples represent impurities that could affect results of mRNA quantification and cDNA synthesis. In most cases, the DNA impurities in RNA samples are removed using enzyme deoxyribonuclease (DNase), which specifically breaks down DNA. In order to avoid the addition of DNase into the analyzing sample, the use of immobilized DNase on solid support is recommended. Because of the DNA size, very few supports available on the market enable efficient interaction between immobilized enzyme and DNA.

In recent years a new group of supports named monoliths was introduced. Because of enhanced exchange between mobile and stationary phase separation and bioconversion processes are significantly accelerated. Therefore also the efficiency of DNA removal using immobilised enzyme might be competitive to the degradation with free enzyme.

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2000

Strains of the anaerobic bacterial genus are thought to play an important role in fiber degradation. sp. Mz5 was previously isolated from the rumen of a black and white Friesian cow and its xylanolytic activity was proved to be at least 1,65 times higher than the activities of all of the compared well known xylan-degrading rumen bacterial species and strains (1). High xylanolytic activity was the reason for partial isolation of its xylanases in order to study their special characteristics and possible biotechnological applications later.

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1999

High performance membrane chromatography (HPMC) proved to be a very efficient method for fast protein separations. Recently, it was shown to be applicable also for the isocratic separation of plasmid DNAconformations. However, no study about the separation of small molecules was performed until now. In this work, we investigated the possibility of gradient and isocratic separations of small molecules with Convective Interaction Media (CIM) disks of different chemistries. We proved that it was possible to achieve efficient separations of oligonucleotides and peptides in the ion-exchange mode as well as the separation of small hydrophobic molecules in the reversed phase mode. Fairly good separation of four oligonucleotides could be achieved on the disk of 0.3 mm thickness. The effect of the gradient parameters on the resolution in the case of gradient mode was studied and compared with the separation under isocratic conditions.

It was shown that similar peak resolution can be achieved in both gradient and isocratic modes. In addition, it was found that the flow rate does not have a pronounced influence on the resolution in the flow rate range between 1 and 10 mL/min. However, it seems that the resolution with the flow rate even slightly increases as a consequence of the increased pore accessibility. In accordance with conventional particle HPLC columns, the resolution increases with the monolith thickness. On the other hand, the mobile phase composition has to be carefully adjusted to obtain optimal resolution, especially in the case of isocratic separations. Because of this feature, CIM monoliths seem to be competitive to other, commercially available stationary phases.

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Organic acids are important metabolites of several biochemical pathways in microorganisms and as such they are frequent main or by-products in different bioprocesses. Consequently, a demand for their monitoring is often present. One of the most applied methods for organic acids determination is certainly HPLC using different separation mechanisms such as reversed-phase, ion-exchange or ion-exclusion chromatography, all based on separation under isocratic flow conditions. To achieve the isocratic separation, multiple steps of adsorption-desorption process are needed and therefore conventional chromatographic columns with long layer of separation material were considered as a necessary tool for achieving this effect.

Recently, it was shown that isocratic separation could also be performed on thin monolithic layers. The isocratic separations of plasmid DNA conformers (1), oligonucleotides (2, 3) and peptides (3) in the ion-exchange mode were demonstrated as well as isocratic reversed-phase separation of a mixture of steroids was obtained (3) all on thin GMA-EDMA monoliths commercially available under trademark CIM™ (Convective Interaction Media). The results indicated the possibility of applying CIM™ monolithic columns also for isocratic separation of some other small charged molecules. Since the average analysis time using CIM™ disk monolithic columns is up to a few minutes, these supports can be a material of choice for separation of organic acids.

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CIM® supports are novel monolithic chromatographic supports. In contrast to conventional particle based chromatographic supports they consist of a single porous polymer. The pores form a highly interconnected network, which enables the flow of the mobile phase through the monolith. Molecules to be separated are transported to the surface by the convection. Since the diffusion is not a bottleneck any more, also the resolution and the dynamic capacity of the monolith are flow independent and an average analysis time is typically below one minute. Furthermore, CIM® columns were successfully applied for the purification of proteins directly from the fermentation broth.

Manganese peroxidases (MnP) and lignin peroxidases (LiP) are a family of glicosilated hemo-proteins, which are excreted into the growth medium during the idiophasic growth of the white rot fungus Phanerochaete chrysosporium. They are both involved in the lignin degradation. For their analysis and separation from the growth medium, HPLC is commonly applied. Besides the separation by Na-acetate concentration gradient (2), also the chromatofocusing can be used (3). A fast method for LiP isoenzyme separation from the growth medium of P. chrysosporium using CIM™ QA disk monolithic columns has been recently developed (1). A modified method was tested on the growth medium containing MnP isoenzymes.

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The aim of our work was to study the direct monitoring and purification of proteins from the fermentation broth using ion-exchange CIM® supports. Therefore, we studied the possibility of monitoring and purifying lignin peroxidase extracelular protein isoforms produced by the fungus Phanerochaete chrysosporium. These isoenzymes which also differ in their catalytic properties are able to partially depolymerize lignin and to oxidise several xenobiotics.

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The white rot fungus Phanerochaete chrysosporium under nitrogen or carbon limitation produces extracellular lignin peroxidases (LiP). They are able to partially depolymerize lignin and to oxidize several xenobiotics (DDT, PCB, PAH, etc.). By HPLC separation and isoelectric focusing multiple molecular forms of LiP have been isolated from the culture filtrate. For the isolation of LiP from the growth medium, mostly the HPLC technique with ion exchange Mono-Q or DEAE columns is used. The medium should be dialyzed before separation and usually also concentrated. Medium freezing is used to remove mucilaginous polysaccharides which disturb separation. The whole procedure is time consuming and information about isoenzyme content and their relative amounts in the growth medium is delayed for at least 1 day. HPLC separation itself lasts nearly an hour. For the separation of LiP isoenzymes from the culture filtrate, we used the monolithic stationary phase with weak (DEAE-diethylamine) and strong (QA-quaternary amine) ion exchange groups commercially available under trademark CIM (Convective Interaction Media). CIM supports are glycidyl methacrylate based monolithic porous polymer supports. As such they differ from conventional particle shaped chromatographic supports. The liquid is forced to flow through the support channels. Molecules to be separated are transported mainly by convection resulting in travelling times shorter for at least an order of magnitude. As a consequence the resolution as well as the binding capacity remain unaffected with the flow rate and a shorter analysis time can be achieved. This effect is even more pronounced in the case of large molecules such as proteins, which have a low diffusion coefficient. As such, CIM units can be advantageous also for lignin peroxidase isoenzymes separation and purification.

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1998

White rot fungus Phanerochaete chrysosporium produces under nitrogen limitation extracellular lignin peroxidases (LiP). They are able to partially depolymerize lignin and to oxidise several xenobiotics (DDT, PCB, PAH,…) and synthetic dyes. Trough HPLC separation and isoelectric focusing multiple molecular forms of LiP have been determined and isolated from the culture filtrate. Depending on growth conditions, separation technique, strain employed and culture age 2-15 different LiP izoenzymes were observed in culture media of Phanerochaete chrysosporium. They are structurally similar but differ in stability, quantity and in catalytic properties. For the isolation of LiP from growth medium, mostly the procedure employing HPLC ionexchange columns as shown on Scheme 1 is used. For the separation of LiP isoenzymes from the culture filtrate, we used CIM (Convective Interaction Media) units. Their advantage is very fast separation of macromolecules due to their particular threedimensional structure. In contrast to particle supports containing closed pores, CIM units consist of monolith porous material containing flow through pores. Therefore, macromolecules to be separated are transported to the active site by convection rather than by diffusion. As a consequence, the separation resolution and dynamic binding capacity are flow independent. As such CIM units can be advantageous also for lignin peroxidase isoenzymes separation and purification.

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