Posters

Membrane based anion exchangers are being used increasingly for purification of monoclonal antibodies. The transition from particle-based anion exchangers is driven partly by the convenience of membranes and partly by the cost saving associated with their disposability, however the feature that makes them functionally superior is more effective mass transport.

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.

Commercially available CIM® disk monolithic columns are intended for very fast analyzes and laboratory purification. Their shape is a compromise to achieve acceptable resolution and binding capacity what make them suitable for wide range of laboratory applications. Separations of complex protein mixtures can be carried out within just a few seconds because of flow unaffected resolution and, on the other hand, purification can be effectuated with high productivity due to flow-unaffected dynamic binding capacity [1]. However, in many cases in the field of molecular biology, only a limited amount of sample is available. In such a case it is beneficial to work with small columns having high resolution or they can be used as affinity columns or bioreactors saving significant amount of valuable ligand. Having this goal in mind we developed CIM® disks with the volume of 1/10th and 1/100th of original volume. In comparison to conventional CIM® disks, they exhibit higher resolution and lower limit of detection, therefore smaller concentrations of target macromolecules can be detected. The separation ability and the protein capacity were tested on anion and cation exchange 3.4 mL and 34m L mini disk monolithic columns.

Analysis of a large numbers of samples requires chromatographic supports that not only enable fast separation and purification of a target biomolecules from a complex matrix but are also involved in an automation process. The 96 – microtiter plate format enables both. Although they are routinely used for decade's only recently few reports about the microtiter plates bearing monoliths as a separation media, were reported [1]. Because of advantageous properties such as flow unaffected dynamic binding capacity and resolution 96 - microtiter plates with methacrylate based monolith were prepared. Characterisation of such plate demonstrated that uniform flow rate can be achieved through all wells and no leakage is present. Efficient separation of proteins was achieved within minute. Furthermore CLC (Conjoined Liquid Chromatography) concept [2] originally derived for analytical columns on CIM disk, can easily be extrapolated to microtiter plates. We demonstrated that multidimensional chromatography with 96 – well plate is feasible and can further accelerate screening processes.

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.

The analysis of molecular interactions is a key part of the drug discovery process, and analytical techniques are available for studying in vitro the ligand/target complex since the early stage of the drug development process.

With regard to the assessment of the activity of chemical libraries, the affinity chromatography on HPLC immobilized-enzyme column (or immobilized enzyme reactors, IMER) is one of most promising methodologies for HTS applications.

Human recombinant acetylcholinesterase (hAChE) represents a well-known target for drug-discovery in Alzheimer’s Disease.

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).

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.

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.

Affinity chromatography is a key method for protein purification. Its main advantage is in the high specificity which enables purification of a single protein from complex biological mixtures. For practical use the specific ligand should be immobilised on insoluble matrix. As a matrix, standard chromatographic supports are commonly used. They are normally in form of small (some m in diameter) particles containing pores to provide high specific surface resulting in high binding capacity. The pores are normally closed on one side, thus the liquid inside them is stagnant and the molecules are transported to the active site by diffusion. Since the diffusion coefficients for macromolecules, such as proteins, are very low, diffusion determines the overall process dynamics. As a consequence, separation or purification of the proteins takes normally 0.5 to 1h even on analytical scale.

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.

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.

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.

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.

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.

Tissue plasminogen activator (t-PA) is serine protease which converts plasminogen into plas-min dissolving the major component of blood clots, fibrin. So, it can be extremely useful in clinical practice to help curing of heart attack victims. The most available way protein producing is genetic engineering where separation and purification of goal protein are one of the important steps in protein producing process.

Recently developed High performance monolithic disk chromatography, HPMDC, seems to be a very attractive way for study quantitative affinity parameters of recombinant proteins with different ligands as well as for protein separations and purifications. High process speed prevents the denatura-tion due to temperature and solvents influence. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting.

It is known that plasminogen, which is the natural substratum for t-PA, can be successfully used as affinity ligand to separate t-PA from cellular media. However, the use of synthetic ligands for affinity chromatography is more preferable due to their higher stability and lower total cost.

The availability of sufficient quantities of quality DNA is always a crucial point in DNA based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods for detection of genetically modified food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3]. The existing methods for DNA isolation from food cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anion-exchange, ion-pair reversephased, and slalom chromatography. Of these, anion-exchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].
Anion-exchange CIM® (Convective Interaction Media) monolithic columns allow fast and flow unaffected separation of several biomolecules, including nucleic acids [5].

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.

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.

The availability of sufficient quantities of quality DNA is always a crucial point in DNA based methods, i.e. for PCR, DNA sequencing, Southern blotting, and microarrays [1]. The same is true for the PCR-based methods for detection of genetically modified food [2]. During the production chain foods passes several physical, biological, and chemical processes, which all negatively influences on the quantity of available DNA. The phenomenon is especially expressive when high temperature treatment is performed at low pH [3].

The existing methods for DNA isolation from food cannot always fulfill the expectations of quantity and quality of isolated DNA. Furthermore they usually include 100 mg of sample and are difficult to scale-up [4]. Four major chromatographic modes are used for the separation of DNA: size-exclusion, anion-exchange, ion-pair reversephased, and slalom chromatography. Of these, anion-exchange chromatography combined with micropellicular packing is described as the most prominent technique so far [1].