Posters

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 for detection of genetically modified 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, anion-exchange, ion-pair reversephased, and slalom chromatography. Of these, anion-exchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].

Plasmids are episomes that have been recognized in few eukaryotic and most prokaryotic species. Some plasmids are excellent genetic vectors and they have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years plasmids were intensively used for gene therapy purposes (1).Most often purification starts with the cells harvest followed by alkaline lysis step in which ribonucleaseA (RNase) is typically used. After that plasmid DNA can be precipitated and used directly or can be further purified by different methods (2).Currently, several chromatographic methods, such as ion-exchange, size exclusion, affinity, and hydrophobic chromatography, have been demonstrated in plasmid purification (3). Until now a limited number of small scale purification methods without use of RNase were published. Convective Interaction Media CIM®is a monolithic chromatographic support for which has been shown that is very efficient for the separation of large molecules, such as proteins, DNA and viruses (4).

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].

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.

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®).

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.

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.

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.