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2004

Immobilized Metal-Affinity Chromatography (IMAC) is a separation technique primarily intended for the purification of proteins with exposed histidine tags. Technique uses covalently bound chelating compounds on chromatographic supports to entrap metal ions, which serve as affinity ligands for various proteins. Iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), carboxymethylated aspartic acid (CM-Asp), and N,N,N’-tris(carboximethyl) ethylenediamine (TED) are chelating compounds, most often used to entrap metal ions, such as Cu2+, Ni2+, Zn2+, Co2+ etc.

Convective Interaction Media CIM® is a monolithic support, which provides high rates of mass transfer at low pressure drops. It has been shown that CIM® supports are very efficient for the separation of large molecules, such as proteins and DNA (1). Recent publication has proved that CIM IMAC column can be used for separation of histidine containing peptides (2). Since efficient separation of large molecules is one of the main advantages of CIM® support, purification of His-tagged recombinant proteins on CIM IMAC column should be not only feasible but also simple, fast and efficient.

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Membrane bound heterotrimeric guanine-nucleotide proteins (G-proteins) are the important components of the cellular signal transduction cascade. They are GTPases which cycle between an inactive and an active configuration by catalysing the exchange of GTP for GDP bound to G subunit. In our study we investigated separation of high affinity GTP'S binding proteins (G-proteins) from plasma membrane of porcine brain by HPLC using CIM® (Convective Interaction Media) supports. CIM® supports proved to be an efficient tool for cytosolic protein separation on second or minute time scale. No study of separation of membrane bound proteins by CIM® supports have been done so far.

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Traditionally, viruses are purified by time consuming methods such as CsCl density gradient centrifugation or similar. These methods are often inefficient and limited to small scale. In recent years different methods for virus purification, based on ion exchange, gel filtration and affinity chromatography have became popular. Recently, CIM® disk monolithic columns were used for successful concentration of two plant viruses (1) and for improved detection of two human viruses (2). Cucumber mosaic virus (CMV) and Tomato mosaic virus (ToMV) were concentrated and subsequently detected from extremely diluted samples in which they were initially undetectable. Successful concentrations of both viruses encourage us to explore the possibilities of CIM® supports for virus purification. As a model virus ToMV was selected. ToMV is a rod shaped plant virus with a typical size of 300 x 18 nm and isoelectric point at pH 4.6.

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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].
Anion-exchange CIM® (Convective Interaction Media) monolithic columns allow fast and flow unaffected separation of several biomolecules, including nucleic acids [5].

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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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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 ribonuclease A (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).

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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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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].

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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).

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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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2001

CIM Convective Interaction Media® are polymer-based monolithic supports which were introduced for chromatographic analyses, in-process control, solid phase extraction, and purification of target biomolecules, both on an analytical and on a preparative scale 1, 2. CIM supports perform high-resolution separations within seconds. This is predominantly due to the convective mass transport of the biomolecules between the mobile and stationary phases and the very low dead volume of the separation unit. One of the main concerns in the last few years was the batch-to-batch reproducibility of the monoliths during manufacturing and the possibility of using the monolithic supports for validated analytical methods. The batch-to-batch reproducibility in product preparation as well as its stability during analytical work should fulfill all the requirements for a validated analytical method. To demonstrate that this is possible, we have selected one complex example – the determination of impurities in immunoglobulins (IgGs) where a multidimensional, so called CLC (Conjoint Liquid Chromatography), approach combining the ion exchange and affinity chromatography was needed to properly analyze the sample.

Therefore, two CIM Protein G disks and one CIM QA disk were placed in series in one housing. Binding conditions were optimized in a way that the IgGs were bound to the CIM Protein G disks while Transferrin and Albumin were separated on a CIM QA disk. A complete separation of all three proteins was achieved in five minutes.

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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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Convective Interaction Media (CIM) are newly developed polymer-based monolithic supports which were introduced for chromatographic analyses, in-process control, solid phase extraction and laboratory purification of target biomolecules, both on analytical and on preparative scale. CIM supports allow high resolution separations which can, in case of analytical units - disks - be carried out within seconds (Figures 1 and 2). This is due to predominantly convective mass transport of biomolecules between the mobile and stationary phase and low dead volumes. Additionally, the dynamic binding capacity is not affected by high flow rates.

CIM can be scaled up to preparative level. For this purpose, the tubular-shaped monolithic units are prepared and placed in special housings (Figure 3). These preparative tubes are intended for very fast preparative purification of biomolecules from complex mixtures. Due to their special design, which allows radial flow of the liquid through the porous wall of the tube, and due to their low resistance to flow, the separations can be carried out at high flow rates and low back pressures (Figure 4). Small-scale preparative tubes are made of the same material as analytical CIM disks. In this way, the purification and monitoring processes can be performed on the same type of support by applying identical separation modes. The scaling-up from analytical to preparative level can therefore be carried out in a much shorter time, thus considerably reducing the cost of process development. In addition, this speed has an economic potential not only for faster and therefore cheaper production, but it will also lead to better quality and yield of unstable products.

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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 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 [1]. 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 [2]. This paper describes the use of a CIM® Convective Interaction Media [3] for fast in-process analyses and preparative separations (up-scaling) of pharmaceutically relevant biopolymers such as clotting factor IX. Human factor IX is a vitamin K-dependent multidomain glycoprotein synthesized in liver [4]. The absence or a defect of factor IX causes haemophilia B, a genetic disease in which the clotting cascade is disturbed. The concentration of factor IX in human plasma is about 5 μg/ml (0.1 μM). Because of the low concentration in human plasma, isolation of clotting factor IX has been performed by a combination of different chromatographic methods. However, it has not been possible to remove vitronectin, one of the final contaminants from factor IX purified with conventional gel supports used in the manufacturing process of commercial factor IX preparations. This paper investigates the application of CIM® monolithic columns for the separation of vitronectin from factor IX and fast in-process control of factor IX [5].

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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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