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2008

During last decades different methods for purification of influenza viruses have been described. Most of these methods were developed for purification of egg derived influenza virus which is still the main production system for influenza vaccine viruses. Since cell culture based technology is gaining more and more importance, the need for alternative, efficient and scaleable purification methods has risen. Chromatography is becoming a method of choice for purification of viruses. Relevance of this technique was recently demonstrated also for influenza viruses. Methacrylate monoliths are characterized by large channel diameter, high surface accessibility and convective mass transport. As a consequence they have high binding capacity for large molecules, enable high flow rates at low pressure drop and therefore increase productivity. Recently it has been proven that methacrylate monolithic columns can also be used for purification and concentration of different viruses.

It was the purpose of this work to explore possibilities for purification of influenza viruses on ion exchange methacrylate monoliths. Different subtypes of influenza A and influenza B virus were tested employing various ion exhange monolithic columns.

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During the last decade important developments in molecular medicine and adenoviral vector design have been achieved, leading to an increased use of adenoviral vectors in clinical gene therapy protocols. One of the main advantages of the adenovirus is their ability to replicate at high titres in permisive cell lines. The availability of large quantities of adenoviral vector preparations is recognized as an important limitation to pre-clinical and clinical studies. Consequently there is a global focus on large scale production of adenoviral vectors, providing high titres combined with fast, effective and reliable purification methods.

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2007

A number of IgM monoclonal antibodies are currently in development for treatment of autoimmune disease, infectious disease, and cancer. Growing interest in these molecules has created a need for an accurate, rapid, simple analytical method to measure IgM levels in cell culture supernatants, and to document the distribution of IgM and protein contaminants in chromatography fractions. High performance protein A columns are used for this application with IgG monoclonals, but IgMs are easily denatured by the harsh conditions required for elution of most affinity ligands. However, IgM monoclonals often exhibit strong retention on either cation exchangers, or anion exchangers, or both, making ion exchange chromatography a potential candidate for this application.

The large size of IgMs makes them a major challenge to particle-based chromatography media. Pentameric IgM has a mass of about 0.96 Md, and hexameric IgM about 1.15 Md. Their diffusion constants are about 2.5 x10-7 cm2/sec, about twice as slow as IgG. Since particle-based chromatography media mostly rely on diffusion for mass transport, both resolution and capacity are im- Figure 4 illustrates a modified anion exchange gradient configuration for monitoring the amount of IgM expressed in cell culture supernatants. A wash step was introduced to better remove con- paired, and increasingly so at higher flow rates.

Monolithic ion exchangers are characterized by an interconnected system of channels with diameters ranging 0.5 to 2.0 microns. This pore architecture supports convective flow, which conserves high resolution at high flow rates.[1] The lack of a void volume removes the major source of dispersion in chromatographic systems. This contributes to sharper peaks, which improves both resolution and sensitivity. Capacity is also conserved at high flow rates. This permits use of a microcolumn format that minimizes assay time and buffer consumption. This combination of features should make monoliths effective analytical tools for IgM.

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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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The rapidly growing interest in the area of proteomics induces intensive efforts to find robust, automated and sensitive high-throughput analytical tools. In this context, the concept of solid-phase digestion (ex. trypsin immobilization on a solid support[1]) has received great attention in the last years. Trypsin (EC 3.4.21.4) has been covalently immobilized on different monolithic supports and resulting bioreactors used as immobilized enzyme reactors (IMERs) for on-line digestion, peptide separation and peptide mapping. Bioreactors efficiencies were evaluated with different recombinant proteins after on-line digestion. The technique used for the separation and identification of peptides was high-performance liquid chromatography coupled with electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS).

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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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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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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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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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