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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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. 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 tubularshaped monolithic units are prepared and placed in special housings. 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. 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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Found recently serine protease called, as tissue plasminogen activator (t-PA) is able to dissolve efficiently the blood clots. Thus this protein seems to be extremely useful in clinical practice in the cases of heart attack victims.

Real process of fibrinolysis in human blood system represents very complicated network of simultaneous biological events. It is clear that t-PA has a branched set of functional complements with their own, and probably different, affinity to this enzyme. It seems to be possible and quite interesting to investigate all these pairs separately creating them in vitro. At the same time, it is clear that the affinity chromatography approach could become as the most convenient way to create such biological pairs.

The recently developed High Performance Membrane (Monolith) Chromatography (HPMC) is quite promising in this regard, because of its high capacity and selectivity, combined with low backpressure and short operation times. Due to the inherent speed of the isolation it facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences.

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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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Convective Interaction Technology (CIM®) offers a number of benefits for the purification of large molecules in comparison with conventional chromatography. The innovative matrix, cast as a single homogeneous piece, means that monolithic columns have a high pressure tolerance and allow fast operating flow rates.

Because the matrix structure is composed of large pores, mass transfer is essentially convective in contrast to conventional chromatography beads, where mass transfer is essentially diffusive. Therefore, CIM can be used at high flow rates without compromising binding capacity.

For these reasons, a monolithic column with anion exchange properties (CIM® QA) was selected to purify a very large protein (8 Mega Dalton) extracted from a marine mollusc.

Because 150 g of protein was required to perform preclinical trials, a scale-up of the process had to be designed and implemented. Early stage process development was carried out on an 8 mL column to determine the column loading capacity as well as the yield and the process reproducibility.

To improve binding on the column, stabilising agents had to be removed prior to this purification step. The protein had been observed to precipitate within hours of the removal of these reagents. Therefore, a suitable time frame for protein processing had to accommodate this instability.

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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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CIM (Convective Interaction Media) represent a new generation of chromatographic supports. In contrast to conventional particle supports, where the void volume between individual porous particles is unavoidable, CIM supports consist of a single monolith with open channels. In this way, molecules to be separated are transported into the pores by convection, resulting in short separation times.

CIMsupports proved to be very efficient for extremely fast separations of proteins in ion exchange, hydrophobic interaction and affinity chromatography mode. Recently, the successful separation of DNA as well as some smaller molecules like e.g. peptides and oligonucleotides were also performed.

All the above mentioned separations were carried out on an analytical scale with the use of 0.34 mL CIM discs. The scale-up of monolithic units was limited mainly due to the problems associated to the mechanical stability, poor sample distribution and higher backpressures. The change from the axial to radial flow enables the design of the so-called 8 and 80mLCIM tubes. They were basically designed for very fast purification of macromolecules.

In this work we present some basic characteristics of these newly developed units in terms of separation and binding capacity. In addition, some practical examples will be given and discussed as well.

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Isolation and purification of proteins, peptides and polynucleotides as well as fractionation of biological mixtures are of great importance both for the solution of theoretical problems in chemistry and biology and the
realization of practical plans connected, in particular, with the production of medicines on the basis of large biomolecules. An important problem in the production of biological substances for medicine is to work out the step of their isolation and fine purification, e.g. creation of high performance separation methods, particularly, the chromatographic techniques. Here, fast and efficient affinity separations based on dynamical interaction of biocomplements play very important role.

High Performance Membrane (Monolith) Chromatography (HPMC) allows to solve all problems of High Liquid Chromatography (HPLC) demonstrating a number of number of distinct advantages. A small thickness of separation layer and opened structure of throughput channels where the separation takes place cause minimum difussion resistance for normal mass transport of the substances as well as low working back pressure and thus, the possibility of use of high elution flow rates.

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In order to enable the detection of low abundance proteins from human plasma, it is necessary to remove high abundance proteins. Among them, human serum albumin and immonoglobulin G represent more than 75 % of all abundance proteins. There are many strategies developed for an efficient removal of these two main proteins, the majority of them rely on highly selective, yet expensive affinity techniques. In this work an affinity monolithic column was used for the depletion of IgG. For the removal of HSA we tested an alternative - complementary approach, where an ion-exchange mode was used as one of the depletion steps. the results were compared to the ones obtained by by using the prseudoaffinity columns.

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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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