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2003

The availability of sufficient quantities of quality DNA is always a crucial point in DNA-based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods of GMO detection in food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3].

The existing methods, for DNA isolation from food, cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anionexchange, ion-pair reverse-phased, and slalom chromatography. Of these, anionexchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].

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2002

The progress in gene-therapy and DNA vaccination leads to a growing demand of therapeutic applicable plasmid DNA (pDNA). To guarantee the supply for the clinical trials and finally for the market new pDNA production processes, which meet all regulatory requirements, have to be developed. Conventional small scale techniques can not easily be transferred to the manufacturing scale (technical reasons and safety considerations). We developed a generic large scale process for highly purified plasmids “free” of bacterial contaminants which works without enzymes, detergents (except SDS during the cell lysis) and organic solvents.

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Most commonly plasmids are manufactured by fermentation of E. coli. In the cells several isoforms of the plasmid are generated: supercoiled (sc), open circular (oc) and linear as well as dimeric forms. After alkaline lysis plasmids are accompanied in solution by genomic DNA (gDNA), RNA, proteins and other cell compounds [1]. In addition to these impurities, the plasmid isoforms have to be separated efficiently in order to get a final product containing > 95 % of ccc form [2]. Chromatographic resins used in biotechnology are usually designed for the separation of polypeptides, providing only low capacity for polynucleotides (< 1 mg/mL).

In this work we present an optimised purification step for large scale purification of therapeutic applicable pDNA, based on an alternative chromatography resin (CIM Convective Interaction Media®).

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2000

Production and downstream processing in biotechnology requires fast and accurate control of each step in the process. Liquid chromatography of biopolymers on so-called soft supports is typically slow, often causing significant product degradation. One way of improving these boundary conditions in liquid chromatography is the use of monolithic adsorbents. The basis for fast separations with such media is a reduced mass transfer resistance owing to the fact that pore diffusion is practically non-existent. Chromatography with compact, porous units such as monolithic columns is being used increasingly for analytical and preparative separations of biopolymers with apparent molecular mass ranging from several thousand to up to several million.

This paper describes the use of a CIM® Convective Interaction Media for fast purification of plasmid DNA as well as for the concentration of viruses. Plasmid DNAs are circular duplex DNA molecules that are maintained stable as episomal genetic information within bacteria. They play an important role in gene technology - they are used for applications such as transformation, sequencing, transfection studies, etc. These applications require satisfactory purity of used plasmid DNA. For purification of plasmid DNA from Escherichia coli, monolithic units as anion-exchangers (CIM® DEAE and QA disks) were used. Separation of RNA from DNA as well as concentration of plasmid DNA were performed on the same disks.

All the methods for concentration of viruses, in general, are expensive, time-consuming and they are frequently not very successful. Therefore an attempt to bind viruses on an anion exchanger (CIM® DEAE disk) and elute bound virions in small volume (concentration) was done. As a model virus, measles was chosen. Using CIM® DEAE disk concentration of the measles viruses was successfully performed in less than 10 minutes.

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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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Production and down-stream processing in biotechnology requires fast and accurate control of each step in the process. Liquid chromatography of biopolymers on so-called soft supports is typically slow, often causing significant product degradation. One way of improving these boundary conditions in liquid chromatography is the use of monolithic adsorbents. The basis for fast separations with such media is a reduced mass transfer resistance owing to the fact that pore diffusion is practically non-existent. Chromatography with compact, porous units such as monolithic columns is being used increasingly for analytical and preparative separations of biopolymers with apparent molecular mass ranging from several thousand to up to several million.

This paper describes the use of a CIM® Convective Interaction Media for fast purification of plasmid DNA as well as for the concentration of viruses.

Plasmid DNAs are circular duplex DNA molecules that are maintained stable as episomal genetic information within bacteria. They play an important role in gene technology - they are used for applications such as transformation, sequencing, transfection studies, etc. These applications require satisfactory purity of used plasmid DNA. For purification of plasmid DNA from Escherichia coli, monolithic units as anion-exchangers (CIM® DEAE and QA disks) were used. Separation of RNA from DNA as well as concentration of plasmid DNA were performed on the same disks.

All the methods for concentration of viruses, in general, are expensive, time-consuming and they are frequently not very successful. Therefore an attempt to bind viruses on an anion exchanger (CIM® DEAE disk) and elute bound virions in small volume (concentration) was done. As a model virus, measles was chosen. Using CIM® DEAE disk concentration of the measles viruses was successfully performed in less than 10 minutes.

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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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Synthetic oligonucleotides play an important role as novel therapeutic agents.

One of the most important, but also very time-consuming steps in synthetic oligonucleotides production is their purification. Due to their high-resolution power, reversed-phase and ion-exchange chromatography are the most widely used techniques for these purposes. For the reversed-phase separations oligonucleotides need to be kept as 5'-O-dimethoxytrityl derivatives until the purification process is completed and only then the detritylation takes place. Both these steps lower the yield of the production process. In the contrary, ion-exchange chromatography offers applications to deprotected oligonucleotides directly and that is the reason why this chromatography mode is more preferred.

Convective Interaction Media (CIM) are newly developed polymerbased monolithic supports allowing high resolution separations which can be carried out within seconds in the case of analytical units - disks. This is due to predominantly convective mass transport of biomolecules between the mobile and stationary phase and very low dead volumes. Additionally, the dynamic binding capacity is not affected by high flow rates.

In this work weak (DEAE) anion-exchange CIM supports have been successfully applied for the analysis and purification of synthetic oligonucleotides.

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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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