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2007

IgM can be used for several purposes such as early detection of certain diseases or, when labelled, localized cancer tumours. For their purification commonly chromatography is used. Methods for purifying such big molecules (M.w. around 950 kDa) are usually long and time consuming since these molecules have extremely low mobility therefore mass transfer between mobile and stationary phases is significantly reduced. When purified using affinity mode, serious decrease in IgM activity can occur because of long exposure to low pH in which they are unstable. Furthermore, because of their size, the IgM capacity of convenctional resins is rather low. CIM monoliths were already successfully used for fast separation of large molecules. In this work we tested applicability of anion-exchange CIM monolithic columns for preparation of IgM.

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2005

The Inter-alpha inhibitor protein family is comprised of complex plasma proteins that consist of a combination of multiple polypeptide chains (light and heavy chains) covalently linked by a chondroitin sulfate chain. The major forms found in human plasma in high concentration are Inter-alpha inhibitor (Ial), which consists of two heavy chains (Hl & H2) and a single light chain, and Pre-alpha Inhibitor (Pal), which consists of one heavy (H3) and one light chain (Fig 1). The light chain (bikunin) is known to inhibit several serine proteases, such as trypsin, human leukocyte chistase, plasmin and cathepsin G which are involved in inflammation, sepsis, tumor invasion and formation of metastasis. Recently, a monoclonal antibody against human inter-alpha inhibitor proteins (MAli 6931) was developed in our laboratory.

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Immobilized Metal-Affinity Chromatography (IMAC) is a chromatographic separation technique primarily used for the purification of proteins with exposed histidine residues and for recombinant proteins with histidine tags. Technique uses covalently bound chelating compounds on chromatographic supports to entrap metal ions, such as Cu2+, Ni2+, Zn2+, Co2+, which serve as affinity ligands for various proteins. CIM Convective Interaction Media is a monolithic chromatographic support intended for separation of large biomolecules, such as proteins, DNA and also viruses.

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Immobilized Metal-Affinity Chromatography (IMAC) is a separation technique primarily intended for the purification of proteins with exposed histidine tags. Technique uses covalently bound chelating compounds on chromatographic supports to entrap metal ions, which serve as affinity ligands for various proteins. Iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), carboxymethylated aspartic acid (CM-Asp), and N,N,N’-tris(carboximethyl) ethylenediamine (TED) are chelating compounds, most often used to entrap metal ions, such as Cu2+, Ni2+, Zn2+, Co2+ etc.

Convective Interaction Media CIM® is a monolithic support, which provides high rates of mass transfer at low pressure drops. It has been shown that CIM® supports are very efficient for the separation of large molecules, such as proteins and DNA (1). Recent publication has proved that CIM IMAC column can be used for separation of histidine containing peptides (2). Since efficient separation of large molecules is one of the main advantages of CIM® support, purification of His-tagged recombinant proteins on CIM IMAC column should be not only feasible but also simple, fast and efficient.

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Viruses have proven to be useful vectors for gene therapy purposes. As therapeutics for human use they must be pure and contaminant free. Traditionally, viruses are purified by complicated and time consuming methods such as CsCl density gradient centrifugation or similar. In recent years liquid chromatography has became interesting method for virus purification. It provides high level of purity required for human use and increases productivity. Traditional chromatographic supports were mostly designed for purification of proteins and as such are commonly inappropriate for viruses. Alternative to traditional chromatographic support are methacrylate monoliths (CIM monoliths), characterized by large channel diameter, high surface accessibility and convective mass transport.

The aim of this work was to characterize CIM supports for separation and possible purification of a model virus Tomato mosaic virus (ToMV) from crude plant material.

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A large number of diagnostics and several therapeutic monoclonal antibodies (mAbs) have been approved worldwide and many more are expected to be approved and licensed in the near future. The reality and the fact that purification or downstream processing can contribute up to 80% of the total production costs of a biopharmaceutical, enhance the need for efficient purification methods. Liquid chromatography provide high level of purity required for human use, increases productivity and has therfore become the method of choice for purification of biopharmaceuticals.

Purification of mAbs can be achieved by a number of chromatographic methods, Protein A and Protein G affinity chromatography being especially powerful enabling high product purity with single chromatographic step.

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Human plasma is a rich and readily accessible source for the detection of diagnostic markers and therapeutic targets for various human diseases. These are usually proteins that are present in human plasma in extremely low concentrations and are often masked by the high abundance proteins like immunoglobulin G (IgG) and human serum albumin (HSA), which represent over 75 % of all proteins. In order to enable the detection of potential biomarkers, IgG and HSA should be efficiently removed from the starting sample. In this work an affinity and a pseudoaffinity chromatographic column, used for an efficient removal of IgG and HSA from human plasma, were thoroughly characterized. A CIM monolithic column bearing Protein G ligands was
used for the removal of IgG, and a column bearing an anti-HSA dye was used for the depletion of HSA.

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Fast diagnosis of different infections is a crucial for a successful medical treatment. For diagnosis of certain diseases, separation of IgG and IgM in human serum is required to prevent interference or competing. This is usually achieved by adding adsorbent containing antihuman antibodies to the sample. Incubation from half to one hour is needed to achieve the complete removal of the antibody.

A quicker way to achieve the removal of antibody would be the use of a chromatographic support with specific ligand, which selectively binds the antibody. For example, a Protein G column can be used for removal of IgG. This is faster, but also much more expensivfe way of removing IgG's.

CIM Convective Interaction Media stationary phases represent a novel generation of stationary phases for liquid chromatography. Because of their monolithic structure, being designed for the separation and purification of macromolecules, they exhibit a higher dynamic capacity for very alrge molecules in comparison to traditional stationary phases, combined with much shorter process time that further result in a decreased loss of the biologic activity.

In this work, we present low price ligands (coupled to CIM chromatographic support), which can be used for efficient separation of IgG and IgM antibodies.

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2004

Immobilized Metal-Affinity Chromatography (IMAC) is a separation technique primarily intended for the purification of proteins with exposed histidine tags. Technique uses covalently bound chelating compounds on chromatographic supports to entrap metal ions, which serve as affinity ligands for various proteins. Iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), carboxymethylated aspartic acid (CM-Asp), and N,N,N’-tris(carboximethyl) ethylenediamine (TED) are chelating compounds, most often used to entrap metal ions, such as Cu2+, Ni2+, Zn2+, Co2+ etc.

Convective Interaction Media CIM® is a monolithic support, which provides high rates of mass transfer at low pressure drops. It has been shown that CIM® supports are very efficient for the separation of large molecules, such as proteins and DNA (1). Recent publication has proved that CIM IMAC column can be used for separation of histidine containing peptides (2). Since efficient separation of large molecules is one of the main advantages of CIM® support, purification of His-tagged recombinant proteins on CIM IMAC column should be not only feasible but also simple, fast and efficient.

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Membrane bound heterotrimeric guanine-nucleotide proteins (G-proteins) are the important components of the cellular signal transduction cascade. They are GTPases which cycle between an inactive and an active configuration by catalysing the exchange of GTP for GDP bound to G subunit. In our study we investigated separation of high affinity GTP'S binding proteins (G-proteins) from plasma membrane of porcine brain by HPLC using CIM® (Convective Interaction Media) supports. CIM® supports proved to be an efficient tool for cytosolic protein separation on second or minute time scale. No study of separation of membrane bound proteins by CIM® supports have been done so far.

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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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2001

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.

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2000

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.

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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].

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1999

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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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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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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