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2005

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

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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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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2003

The only four drugs approved for the clinical treatment of Alzheirner’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 acetylcholinesterase (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 we reported the immobilisation of AChE on a silica based chromatographic column (50 x 4.6 mm 1.0.) The yeld of immobilization and the stability of the AChE-IMER were considered satisfactory, but 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 mm I.D.).

CIM® (Convective Interaction Media) monolithic supports (Biaseparations. Lubiana) 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 CIMK disk with immobilised human recombinant acetylcholinesterase (HuAChE-ClM® Disk) was developed. The activity of immohilised enzyme, the long term stability and reproducibility were tested. HuAChE-CIM® 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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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 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

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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Monolithic chromatographic supports can efficiently be used for fast separation and purification of different types of molecules, both in the analytical and preparative scale. CIM Convective Interaction Media™ monolithic columns are macroporous polymeric supports that allow in-seconds separation of proteins and other biomolecules in gradient and isocratic modes.

In this work, the results showing the main characteristics of CIM™ columns are presented. The breakthrough curves at different flow rates were measured and it is shown that the dynamic binding capacity is practically unaffected by increased flow rates. The adsorption isotherm is almost rectangular exhibiting a highly favourable conditions for binding the tested components to the matrix. Furthermore, relatively high binding capacity is still maintained at elevated ionic strengths of the binding buffer. Finally, the HETP values of the components with different molecular masses are presented.

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There are many different chromatographic supports on market. Although main part of them are particle shaped supports, the so-called monoliths are becoming increasingly more important. Particle based supports are commonly uniform-sized of some micron with high porosity. The pores are required to increase the specific surface area and, as a consequence, to increase the binding capacity. Since the pores are closed on one side, the liquid inside them is stagnant and the movement of molecules is governed by diffusion. Therefore, to obtain a good separation and a high binding capacity, low flow rates should commonly be applied. This results in flowdependent resolution of the separation and dynamic binding capacity.

In contrast to conventional porous particles the morphological characteristics of CIM supports are characterised by a single monolithic unit that contains pores, opened on both sides. These pores are highly interconnected forming a flow-through a network. All the mobile phase is forced to run through these open pores, therefore, the mass transfer between stationary and mobile phases is based on convective flow. One of the key features of monolithic units is their pore size distribution that should enable low back pressure at high throughputs together with high specific surface area, needed for high binding capacity.

In this work, dynamic characteristics of CIM disks bearing weak anion exchange groups for binding Bovine Serum Albumin (BSA) were studied. Reproducibility was checked and protein concentration as well as the flow rate were varied. Preliminary results confirm the flow independence of the dynamic binding capacity in the whole range of applied flow rates.

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