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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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2006

Gene therapy has already shown some great results in treatment and cure of some monogene diseases, such as diabetes. While the use of genetically modified viruses raises safety concerns, synthetic formulations of genes inserted in plasmids are regarded as safer. At present, most clinical trials involve plasmids smaller than 10 kb. However, the concern that regulation of the functioning of the gene is ensured together with the expectation of the progression of gene therapy to multigene disfunctions, like cancer or complex nevrodegenerative disfunctions (Alzheimer disease), will require the production of larger plasmids [1].

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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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Plasmids are excellent genetic vectors and have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years, plasmids are intensively investigated for gene therapy purposes and genetic vaccination. In this case, plasmid DNA (pDNA) of high purity is required. To follow such demands, several chromatographic steps are commonly needed. In the case of buffer compatibility, columns can be connected in-line to overcome time consuming and yield lowering multiple chromatographic steps. Since each of the unit operations contributes to the dispersion, the resolution is further decreased by each chromatographic step. This drawback might be surmounted by combining several chromatography steps into a single chromatography column. This approach is known as multidimensional or conjoint liquid chromatography (CLC).

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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

By using a combination of two CIM® tube monolithic columns, OH and DEAE chemistry, we were able to successfully purify plasmid DNA from bacterial culture without using RNase. Purified plasmid DNA is very pure, since common contaminants, such as proteins, genomic DNA, endotoxins and RNA were under the detection limit. The scale up units produced according to cGMP standard are already used for the purification of plasmid DNA for gene therapy purposes on industrial scale.

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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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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

Plasmids are episomes that have been recognized in few eukaryotic and most prokaryotic species. Some plasmids are excellent genetic vectors and they have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years plasmids were intensively used for gene therapy purposes (1). Most often purification starts with the cells harvest followed by alkaline lysis step in which ribonuclease A (RNase) is typically used. After that, plasmid DNA can be precipitated and used directly or can be further purified by different methods (2). Currently, several chromatographic methods, such as ion-exchange, size exclusion, affinity, and hydrophobic chromatography, have been demonstrated in plasmid purification (3). Until now a limited number of small scale purification methods without use of RNase were published. Convective Interaction Media CIM® is a monolithic chromatographic support for which has been shown that is very efficient for the separation of large molecules, such as proteins, DNA and viruses (4).

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Plasmids are episomes that have been recognized in few eukaryotic and most prokaryotic species. Some plasmids are excellent genetic vectors and they have been widely used in gene manipulation and recombinant DNA technology for a long time. In recent years plasmids were intensively used for gene therapy purposes (1).Most often purification starts with the cells harvest followed by alkaline lysis step in which ribonucleaseA (RNase) is typically used. After that plasmid DNA can be precipitated and used directly or can be further purified by different methods (2).Currently, several chromatographic methods, such as ion-exchange, size exclusion, affinity, and hydrophobic chromatography, have been demonstrated in plasmid purification (3). Until now a limited number of small scale purification methods without use of RNase were published. Convective Interaction Media CIM®is a monolithic chromatographic support for which has been shown that is very efficient for the separation of large molecules, such as proteins, DNA and viruses (4).

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2002

The progress in gene-therapy and DNA vaccination leads to a growing demand of therapeutic applicable plasmid DNA (pDNA). To guarantee the supply for the clinical trials and finally for the market new pDNA production processes, which meet all regulatory requirements, have to be developed. Conventional small scale techniques can not easily be transferred to the manufacturing scale (technical reasons and safety considerations). We developed a generic large scale process for highly purified plasmids “free” of bacterial contaminants which works without enzymes, detergents (except SDS during the cell lysis) and organic solvents.

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Most commonly plasmids are manufactured by fermentation of E. coli. In the cells several isoforms of the plasmid are generated: supercoiled (sc), open circular (oc) and linear as well as dimeric forms. After alkaline lysis plasmids are accompanied in solution by genomic DNA (gDNA), RNA, proteins and other cell compounds [1]. In addition to these impurities, the plasmid isoforms have to be separated efficiently in order to get a final product containing > 95 % of ccc form [2]. Chromatographic resins used in biotechnology are usually designed for the separation of polypeptides, providing only low capacity for polynucleotides (< 1 mg/mL).

In this work we present an optimised purification step for large scale purification of therapeutic applicable pDNA, based on an alternative chromatography resin (CIM Convective Interaction Media®).

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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

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.

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