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2014

Biological samples often consist of a main component, such as albumin in serum, and many other constituents, present in smaller quantities, but nevertheless of high importance in biological systems. When detection of the low-abundance molecules is needed, the main component could interfere with the analyte, complicating the analysis or even making it impossible. In such cases a possible approach is to remove the interfering main component from the sample before the analysis.

Monolithic columns (CIM®) are a great foundation to build affinity chromatography methods, as they offer fast flow rates and can be modified to accomodate various ligands. We selected two most promising approaches for oriented binding of antibodies to the monolithic support. One approach was to bind antibodies to a protein A (pA) column with consequent crosslinking of the protein complex. The other approach was to chemically activate antibodies and bind them selectively to hydrazide-modified (HDZ) monolith surface.

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Interactions between antibodies and their antigens are highly selective and therefore immensely popular for affinity chromatography. Consequently, numereous antibody immobilizations were performed on monolithic supports via different activation chemistries in the last decade. Despite the work already done there was no systematic study, where as many as possible activation chemistries were tested for the immobilization of a model monoclonal antibody with subsequent chromatographic characterization of the affinity support. In this work, various preactivated CIM monolithic columns were used for the immobilization of a model monoclonal IgG.

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Monolithic ion exchange CIM® (Convective Interaction Media) columns have been proven in quantitative analysis of different immunoglobulins such as IgM and IgG from human plasma or cell supernatants. The separation mechanism is based on ionic interactions between the ion exchange monolith and immunoglobulin that are controlled by salt concentration. Here we present another possibility of IgM determination based on monolithic CIM® OH columns where the interactions may be controlled by changes in salt concentration or by pH increase. A method for quantitative HPLC determination of IgM in cell supernatant with fluorimetric detection was developed on CIM® OH column (0.34 mL) by means of pH increase. Optimal separation of IgM from cell supernatant matrix was achieved by combining acetate and phosphate buffer in a suitable gradient profile. Two different quantification methods, i.e. calibration curve and standard addition.

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Enrichment of phosphopeptides prior to LC-MS analysis is a crucial sample preparation step because of their low stoichiometry in biological sample, longer retention on reversed phase columns, and lower ionization efficiency compared to non-phosphorylated peptides [1].The use of metal oxides, most prominently of TiO2 enabled efficient and relatively simple phosphopeptide-enrichment. In this study a new monolithic column from BIA Separations containing immobilized TiO2-nanoparticles was tested for its ability to enrich phosphopeptides. The TiO2-column was also tested for possible carryover originating from biological samples. In conclusion, tested monolithic TiO2 columns show significant binding ability for phosphopeptides and are considered as suitable for phosphopeptide enrichment.

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The demand for human immunoglobulin is invariably increasing on an annual basis. To satisfy demands, different manufacturing processes are used to isolate immunoglobulins from human plasma. A quest for alternative paths in manufacturing not only requires development of the most economical manufacturing process, but also a rapid method development and development of reliable analytics for manufacturing monitoring. For an efficient improvement of the purification methods as well as for in-process control during manufacturing stage, the usage of reliable and fast analytical techniques are of crucial importance.

Fast and reliable fingerprint-based method for characterization of immunoglobulin G (IgG) prepared from Cohn I+II+III paste in two chromatographic steps is presented. The fingerprint method bases on partial separation of proteins in linear gradient on CIMac QA 0.1 mL column. Partial separation of proteins does not allow simple quantitative analysis of the samples during the IgG production from Cohn I + II + III paste, however, a very accurate qualitative information about the composition of the sample can be obtained in less than 5 minutes.

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2013

The development of safe, effective, and affordable vaccines has become a global effort due to its vast impact on overall world health conditions. A brief overview of cancer vaccine characterization techniques, especially in the area of high-resolution mass spectrometry, is presented. It is highly conceivable that the proper use of advanced technologies such mass spectrometry, along with the appropriate chemical and physical property evaluations, will yield tremendous in-depth scientific understanding for the characterization of vaccines in various stages of the development. This work presents the physiochemical and biological characterization of two cancer vaccines: Racotumomab and Her1-ECD. Racotumomab monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH)3, in several clinical trials for melanoma, breast, and lung cancer. The Her1-ECD is a vaccine preparation based on the extracellular domain of HER1 and it is being evaluated in Phase I clinical study in patients with refractory prostate cancer.

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2012

Glycosylation is one of nature mechanism for invreasing the diversity of protein structures affecting biophysical vjaracterostocs and bioactivity. Glycoproteins exist as mixture of different isoforms ("glycoforms"). In this mixture a group od different glyco components is attached to individual glycosylation site. Different glyco componets attached to the same site may have diggerent effect on biophysical charachteristics of glycoproteins. The type of glycosylation and the degree of heterogenity are important for many reasons starting from stability, activity, clinical efficency (toxicity, pharmacokinetics, immunogenicity), to standardization and patentability.

Thus, it is necessary to separate glycoforms and as much as possible to difine the heterogenity i.e. population of of glyco components attached to the singele glycosysilation site.

External invertase is a widely usef model for studying the influance of the glyco-component on protein stability. External invertase from yeast Sccharomyces cerevisiae has 14 potential N-glycosylation sites in the sequence, 13 of which are fully or partially glycosylated with olygomannans of varying sizes.

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Extensive research in the last two decades has led to the realization of Immunoglobulin M (IgM) as a potential therapeutic and diagnostic agent for autoimmune diseases, infectious diseases and as an AIDS and cancer vaccine. Growing interest in these molecules has created a need for an accurate, rapid and simple analytical method to measure IgM concentrations during the production (in-process control) in cell culture supernatants as well as in all purification steps in the downstream processing.

Convective interaction media (CIM) monolithic columns has been increasingly recognized as a quantification tool for large molecules. Affinity ligands like protein A and protein G are the most common ligands used for antibody capture and analysis.

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There is an increasing demand for highly purified immunoglobulin G since they have found wide range of potential application in immunodiagnostics and immunotherapy.

Human IgG (hIgG) consists of four subclasses (IgG1, IgG2, IgG3 and IgG4) that show differences in some of their physicochemical characterictics and biological properties.

The present research project aims to separate subclasses of hIgG using monolithic stationary phase by SMB technology.

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2010

Protein L binds certain types of kappa light chains containing Fv and Fab fragments prepared from antibodies. In the case of IgG's the strong binding affinity refers only to human, mouse and rat species. It offers an advantage over Protein A and G as it binds to kappa light chains regardless of heavy chain subclass and can therefore binds up to 60% of IgG antibodies from human serum sample.

The main goal of our work was the preparation and characterization of CIM Protein L disks. First, Protein L disks with different densities of Protein L on the support were prepared in order to define the dependance of the IgG capacity on the amount of the bound Protein L. Further on, the method of characterization of Protein L disk using IgG was developed. In the end, the stability of the developed CIM Protein L disks in different solutions was tested in order to define the operating and storage conditions.

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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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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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