Posters

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.

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.

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.

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.

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.