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2005

The analysis of molecular interactions is a key part of the drug discovery process, and analytical techniques are available for studying in vitro the ligand/target complex since the early stage of the drug development process.

With regard to the assessment of the activity of chemical libraries, the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMER) is one of most promising methodologies for HTS applications.

Human recombinant acetylcholinesterase (hAChE) represents a well-known target for drug-discovery in Alzheimer’s Disease.

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Affinity chromatography is a key method for protein purification. Its main advantage is in the high specificity which enables purification of a single protein from complex biological mixtures. For practical use the specific ligand should be immobilised on insoluble matrix. As a matrix, standard chromatographic supports are commonly used. They are normally in form of small (some m in diameter) particles containing pores to provide high specific surface resulting in high binding capacity. The pores are normally closed on one side, thus the liquid inside them is stagnant and the molecules are transported to the active site by diffusion. Since the diffusion coefficients for macromolecules, such as proteins, are very low, diffusion determines the overall process dynamics. As a consequence, separation or purification of the proteins takes normally 0.5 to 1h even on analytical scale.

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2004

Tissue plasminogen activator (t-PA) is serine protease which converts plasminogen into plas-min dissolving the major component of blood clots, fibrin. So, it can be extremely useful in clinical practice to help curing of heart attack victims. The most available way protein producing is genetic engineering where separation and purification of goal protein are one of the important steps in protein producing process.

Recently developed High performance monolithic disk chromatography, HPMDC, seems to be a very attractive way for study quantitative affinity parameters of recombinant proteins with different ligands as well as for protein separations and purifications. High process speed prevents the denatura-tion due to temperature and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting.

It is known that plasminogen, which is the natural substratum for t-PA, can be successfully used as affinity ligand to separate t-PA from cellular media. However, the use of synthetic ligands for affinity chromatography is more preferable due to their higher stability and lower total cost.

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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

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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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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The only four drugs approved for the clinical treatment of Alzheimer’s Disease (tacrine, rivastigmine, donepezil and galantamine) are acetylcholinesterase inhibitors which act by maintaining high levels of acetylcholine at the muscarinic and nicotinic receptors in the central nervous system. Human acetyicholinesterase (HuAChE) represents a widely studied target enzyme and it is still object of research for the development of new drugs as enzyme inhibitors.

In a previous paper il] we reported the immobilisation of AChE on a silica based chromatographic column (50 x 4.6 mm I.D.) The yield of immobilization and the stability of the AChE—IMEN were considered satisfactory, hut some problems arose. The length of the IMER and the large amount of enzyme covalently bound to the chromatographic support resulted in catalysis product long elution times and some inhibitors aspecific matrix absorption with delayed enzyme activity recovery. In order to avoid these complications and considering the high rate of AChE enzymatic reaction, we decided to reduce the dimension of the solid support for immobilization, hence the amount of immobilized enzyme, by selecting a monolithic matrix disk (12 x 3 min I.D.).

CIMa (Convective Interaction Media) monolithic supports (Bia Separations, Ljubljana) represent a novel generation of stationary phases used for liquid chromatography, bioconversions, and solid phase synthesis. As opposed to individual particles packed into chromatographic columns, CIM supports are cast as continuous homogeneous phases and provide high rates of mass transfer at lower back pressure.

In the present work a CIM® disk with immobilised human recombinant acetylcholinesterase (HuAChECIM€ Disk) was developed. The activity of immobilised enzyme, the long term stability and reproducibility were tested. HuAChECIM disk was applied as an immobilised enzyme micro-reactor (micro-IMER) in on-line HPLC system for inhibitory potency determination of known AChE inhibitors.

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2001

We have developed a screening procedure for peptide ligands for affinity chromatography on the same monolithic support. CIM® monolithic columns used conventionally for analytical and preparative separation of proteins and polynucleotides were minimized to fit into 96 well solid phase extraction plates. Peptide synthesis and screening were performed on the same format using a vacuum manifold for liquid throughput.

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The development of new chromatographic supports with the aim to improve their chromatographic, hydrodynamic and mechanical properties is continually going on.

CIM Convective Interaction Media® monolithic columns represent a new chapter in every mode of the chromatography. Monolithic columns consist of a single piece of a highly porous polymer with a bimodal pore size distribution, forming flow-through channels [1]. Since all of the mobile phase flows through the pores, molecules to be separated are transported to the active sites by convection [2]. Therefore, the entire analysis can be completed in a very short time.

In this work, the performance of novel semi-preparative CIM® RP-SDVB disk monolithic column for separating proteins and peptides has been investigated. Since the column length in the case of gradient separations commonly used for large molecules, does not play a significant role, CIM® RP-SDVB disk monolithic column are extremely short, typically of only 3 mm. The effect of decreasing column length on the resolution under the conditions of a linear gradient has been presented.

Finally, a 1 minute purification of oligodeoxynucleotide from the synthetic mixture has been performed.

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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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CIM® (Convective Interaction Media) supports represent the fourth generation of chromatographic supports monoliths. These glycidyl methacrylate based monolithic columns are commercially available under the name CIM®. In contrast to conventional porous particles the morphology of the CIM® supports is characterised by a single monolithic unit that contains pores, opened on both sides. These pores are highly interconnected forming a flow-through network. Thus the whole mobile phase is forced to run through these open pores, therefore, the mass transfer between stationary and mobile phase is based on convection rather than on diffusion. This transport mechanism enables very fast separations and purifications of components as well as flow-unaffected resolution and dynamic binding capacity. The latter is especially important for the purification of molecules on preparative level where the productivity is essential. One of the key features of monolithic units is their pore size distribution that should enable low backpressure at high throughputs together with high specific surface area, needed for high binding capacity. In this work the effect of different parameters on binding capacity of CIM® monolithic columns is presented.

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There is a rapidly increasing development of new materials in the field of chromatographic supports stimulated by the need to achieve fast and reliable separation of different solutes.

The generally used chromatographic supports are based on beads packed in the columns. Although these stationary phases have been continuously improved over the last decades, there are still some limitations present. The absence of flow within the pore matrix of particles, slow diffusional mass transfer of solutes, in some cases high back pressure and laborious handling represent the major hindrances.

Development of monolithic materials is a chronicle of efforts to overcome problems of packed particles. Monolithic separation media, made in one piece, contain only flow-through pores, which significantly augment the mass transfer based on convection. This enables use of high mobile phase velocities along with low back pressures and therefore fast separations without decrease of resolution.

This report presents the preparation of glycidylmethacrylatestearylmethacryate- ethylenedimethacrylate and styrene-divinylbenzene monoliths. The porous structure of the support obtained will be discussed. Finally, a few examples of separation of proteins and small molecules with monolithic CIM C-18 and CIM SDVB disks in reversed-phase chromatography will be displayed.

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Ion exchange chromatography is one of the most commonly used techniques for the purification and separation of polar samples such as minerals in water or charged biomolecules (Figure 1).

The technique is based upon reversible binding of the charged species to an oppositely charged group that is attached to an insoluble matrix. A quantitative measure of an ion exchanger’s ability to take up exchangeable counter-ions is its capacity, which strongly influence support properties and can be measured by potentiometric titration with a strong acid or base [2]. However, the time to achieve the ion exchange equilibrium (the stationary state the potentiometric titration is based upon) is very long [3]. Consequently, a new method to measure the total ionic capacity of anion exchange resins is being developed.

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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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CIM® disk monolithic columns are monolithic columns based on glycidyle methacrylate ethylene dimethacrylate copolymers. They have become popular for separation of proteins and polynucleotides. A method for directed synthesis of peptides on these monoliths was developed. With a peptide directed against human blood coagulation factor VIII, the functionality of the CIM® disk monolithic column was checked.

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Monoliths are becoming very attractive stationary phases due to their advantageous hydrodynamic characteristics. The main difference in comparison to conventional particle beds is in their structure. Conventional particle based supports consist of few-micrometer sized porous particles while the monoliths consist of a single piece of porous material. The pores are highly interconnected forming a network of channels. Since the flow of the liquid within the channels is driven by the pressure difference, the molecules to be separated are transported to the active sites located on the surface of the channels by convection increasing their mobility by several orders of magnitude. Because of that, it is possible to perform an efficient separation of large molecules within a very short time. Furthermore, the efficiency as well as the dynamic binding capacity are independent on linear velocity within the range of tested flow rates.

Glycidyl methacrylate based monoliths were introduced in 1990. They were polymerised from glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) in the presence of porogens and an initiator. So far they have been successfully applied in a variety of different applications on an analytical scale: for separation and purification of proteins, DNA, smaller molecules like organic acids, hydroxybenzoates, oligonucleotides and peptides as well as sensors incorporated in a FIA system1.

Preparation of large volume GMA-EDMA monoliths is however problematic. The reason is an increase of the temperature inside the monomer mixture during polymerisation since the reaction is highly exothermic. Because of the bulk polymerisation, temperature increase inside the monomer mixture during the polymerisation can not be avoided, resulting in an extremely inhomogeneous structure of the monolith2. In this work, we introduce an approach for the construction of large scale monoliths in the annulus shape and demonstrate their applicability for chromatographic separation and purification.

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1999

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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