Posters

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.

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.

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.

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.

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.

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.

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.

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].

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.