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2017

Production of high value biological therapeutics usually involves complex manufacturing processes with high process variability. Additionally, development of robust and reliable bioprocesses can be challenging. PAT aims to enhance bioprocess understanding and implies a holistic approach to ensure that quality is built into products by design. Efficient PAT therefore calls for fast and robust analytical techniques which enables to asses high quality information about critical quality attributes and key performance indicators as parallel as possible to the manufacturing process. PATfinder™ is unique HPLC system for routine gradient separations that enables every analytical task. Equipped with bio-inert ceramic pump heads is deliberately tailored to meet the demands of analytical applications covering wide range of biomolecules. Highly sensitive and fast multi-wavelength detector enables to detect component peaks even in very fast gradients.

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Preparative scale chromatographic separation of open-circular (oc) from supercoiled (sc) plasmid DNA (pDNA) isoforms has been already established on CIM® C4 with high ligand density (C4 HLD) monolithic columns with sample loading in 3.0 M ammonium sulphate (AS). The process requires high molarity of AS, increasing the overall cost of the process. Sample displacement chromatography (SDC) can be used as an alternative to decrease the AS concentration required during loading onto hydrophobic chromatographic supports. This study compares three chromatographic monoliths with different hydrophobic ligands on the surface (C4 HLD, pyridine and histamine) for the purification of different pDNA vectors in SD mode.

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New vaccines against Influenza A are required each year to keep up with the most virulent evolving strains. This highlights a need for predictive analytical tools that can aid purification process development and validation. Rapid and reliable quantification of Influenza A virus is therefore of the utmost importance for enabling good yields and controlling the costs of the downstream processing. Here we demonstrate the ability of monolithic chromatography media to produce process predictive profiles that can document ability to remove impurities and obtain high product recoveries.

CIMac™ Analytical Columns are short bed high performance monolithic columns offering all the advantages of CIM® monolithic technology. Their small volume and short column length allow the operation at high volumetric flow rates enabling to receive the information about the product quantity and purity in just a few minutes. Hence, the CIMac™ Analytical Columns can be effectively used for the in-process and final control of various samples from different purification process steps.

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2016

Adeno-associated virus (AAV) vectors of various serotypes are considered to have high potential for gene therapy applications. Currently, manufacturing of AAV vectors faces the challenge of co-production of incompletely formed particles lacking a recombinant viral genome. Empty capsids increase the dose of total AAV administered for efficient transduction and are thought to cause unwanted immunological reactions against the virus.Removal of empty capsids during manufacturing, as well as analysis of empty/full AAV particle content is therefore a critical requirement for any AAV production process. This poster demonstrates how CIMmultus™ QA monolithic columns can be used to remove empty AAV capsids from the product chromatographically in a single step.

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Productivity of the downstream bioprocessing depends among others on the efficiency of chromatographic step. One of the crucial chromatographic parameters is dynamic binding capacity (DBC) for certain biomolecule. DBC could be tailored with changing the surface area of convective pores by tailoring the surface of pre-polymerized monoliths using graft or block polymerization of polymer brushes. Grafted CIM monoliths have already been prepared via Radical Polymerization (RP) and successfully characterized (1).

Recently, the implementation and optimization of Controlled Radical Polymerization (CRP) for grafting of large pore monoliths (average diameter 6 μm ) resulted in polymethacrylate-based ionic exchanger with at least 5 times higher DBC compared to non-grafted 6 μm monoliths, while preserving high permeability. The main goal of our study was to chromatographically characterize novel grafted ion-exchanging monoliths (CIM gDEAE and CIM gSO3) to see whether novel columns still retain flow independent chromatographic properties of non-grafted monoliths.

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The upstream and downstream monoclonal antibody (mAb) bioprocessing makes them susceptible to physical and chemical modifications. In the biotechnological production process of mAbs, structural variations may arise due to some enzymatic activity. Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity and cation-exchange chromatography (CEX) is one of the typical approaches for mAb charge variant analyses. We tested several CEX columns under different conditions and the best column for isotype separation was weak cation-exchanging CIMac COOH chromatographic monolith in pH gradient. We have proven a flow independent separation of mAb charge variants and in this way, a resolution comparable to classical CEX particulate-based analytical columns was achieved in only 6 min analysis time.

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To ensure the desired chromatographic characteristics of the CIM® monolithic column at large scales, monolith microstructure morphology, pore size distribution, porosity and surface ligand density should be uniform. To demonstrate the uniformity of large chromatographic monoliths we have developed new testing procedures. By fabricating smaller columns (disks) from different random  positions of larger monolith, non-cGMP compliant chromatographic testing can be applied on the same polymerization batch without affecting the cGMP compliance of large-scale chromatographic monolith. Each individual disk was thoroughly tested and the results were compared to the properties of the large monolith.

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Since plasmid DNA (pDNA) as a pharmaceutical product has stringent requirements of purity and efficacy, one or more chromatographic steps are often used in the downstream processing train. High ligand density butyl-modified (C4 HLD) monolithic support is currently used in a polishing step of a pDNA purification process (1) and is mainly focused to supercoiled (sc) pDNA isoform separation from the open circular (oc) and linear pDNA isoform as well as for removal of remaining gDNA and RNA. The goal of the study was to compare the productivities of two variations of the polishing chromatographic process employing monoliths – classical bind-elute (BE) versus recently described (2) sample displacement purification (SDP). Classical purification requires high concentration of ammonium sulphate (AS) during loading step and elution is then achieved by descending AS gradient. SDP utilises different relative binding affinities of components in a sample mixture and separates pDNA isoforms under overloading conditions, where sc pDNA isoform acts as a displacer of oc or linear pDNA isoform.

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There are many cases, where a single protein needs to be purified from a complex sample. Such proteins manifest themselves as impurities, which can affect further analysis, either by causing specific equipment malfunction or lower yield in the products. In other cases the specific protein is our molecule of interest, for example in glycomics analysis. In both cases high specificity for proteins, reproducibility and reliability is necessary. We have developed a model immunoaffinity column and 96-well plate based on an anti-fibrinogen monoclonal antibody, covalently immobilized onto CIMac™ analytical chromatographic monolith.

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There are many cases, where a single protein needs to be purified from a complex sample. Such proteins manifest themselves as impurities, which can affect further analysis, either by causing specific equipment malfunction or lower yield in the products. In other cases the specific protein is our molecule of interest, for example in glycomics analysis. In both cases high specificity for proteins, reproducibility and reliability is necessary. We have developed a model immunoaffinity column and 96-well plate based on an anti-fibrinogen monoclonal antibody, covalently immobilized onto CIMac™ HDZ analytical chromatographic monolith.

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2015

Methacrylate monoliths (CIM® monolithic columns) allow for very fast and efficient separations and exhibit very high binding capacities for extremely large bio-particles due to their large inner channel diameters and enhanced mass transfer characteristics.
Additionally, the ability to manufacture polymer monolithic materials ranging from analytical to large scale preparative/industrial columns has tremendous advantages. By ensuring the chromatographic properties are consistent over the whole size range, one can easily design and optimize a purification method on laboratory scale and transfer it to a production line with minimal to no additional modifications.

Until now the largest monolithic column had a volume of 8 L, which was large enough to serve the biopharmaceutics' market's needs. Now however, the capacity of that column is already at its upper limit.

By successfully employing the knowledge and experience from almost two decades of monolith production we have managed to overcome the size limitations and polymerize the largest convective chromatographic support made from one piece of material, a 40 L monolithic column.

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CIMac™ r-Protein A Analytical Column is short bed, high performance monolithic column . Primarly is intended for fast, efficient, and reproducible qualitative and quantitative analyses of Immunoglobulin G (IgG). It is suitable for use with HPLC and UPLC systems. Quantification of Immunoglobulin G is possible between 0.2 μg and 20 μg. Its small volume and short column length allow operation at high volumetric flow rates ( up to 3mL/min). The information about product quantity and purity is thus generated in just 1 minute! The column has innovative symmetric design for bi-directional flow, also extending column lifetime.

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Immunoaffinity columns using antibodies as ligands against mammalian membrane proteins could be used for different applications in protein expresion control and, if a standard available, for concentration determination. Additionally these columns are ideal for polishing step of Fc fusion proteins of mammalian receptors.

Most importantly such columns could extract a significant amount of a pure membrane mammalian protein suitable for structural analyses, such as mass spec analysis of their glycans. Immunoaffinity chromatographic monoliths against MULT-1 transmembrane and RAE-1 GPI anchored glycoproteins were developed as a part of Glycomet project with the main goal to analyze the antigen glycan parts.

Two different  preactivated support were used:  hydrazide (HDZ) and carboxy imidazole (CDI).

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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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Surface hydrophobicity/hydrophilicity of chromatographic stationary phases is one of the important characteristics that influence the chromatographic column performance. On the one hand, the surface should be highly hydrophilic to avoid nonspecific adsorption of sample molecules; on the other hand, the hydrophobic surface is crutial to e.g. separate the molecule isoforms.Therefore, fast and easy characterization method to evaluate the surface „hydrophobic/hydrophilic character" could be valuable.

First stage in the development of this method and the objective of this study was to evaluate the hydrophobicity of test set of 1 mL CIM columns with different ligand chemistries and densities. This was achieved by separation of protein mixture under hydrophobic interaction chromatography (HIC) conditions. Proteins were used since monoliths are used mainly in downstream of large biomolecules.

Moreover, since poor recovery under HIC conditions was observed on some columns, the research was additionally expanded with reversed phase chromatography (RPC) to obtain extra information about even more hydrophobic surface properties of monolithic columns. Therefore, after HIC step the RPC step followed and additional elution of proteins was achieved.

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Exosomes are nano-sized vesicles that are released by many different cell types. They are involved in the transport of a wide range of signalling molecules, including mRNA, microRNA and proteins. Exosomes have been found into body fluids and multiple roles have been ascribed to exosomes, in particular in cell signalling where it has been demonstrated their correlation to disease progression and their overexpression as specific tumour cell biomarkers, suggesting their important role in their diagnosis.

This initial screening oriented towards the separation of exosomes from a cell culture supernatant, has been developed by BIA Separations in collaboration with Exosomics Siena. Exosomes used for this study were cultivated in two different cell lines, MeWo and LNCap, and, after the harvesting, a relatively pure target molecule was obtained after several centrifugations, filtrations and batch affinity capture step with a commercial purification kit. In order to speed-up the process and bring current DSP on a higher level, a novel purification approach based on chromatography, using CIM® monolithic columns was investigated. Monolithic supports represent a new generation of chromatographic media. Due to their large inner channel diameters and enhanced mass transfer characteristics, methacrylate monoliths offer efficient and fast separation of large biomolecules like vescicles, pDNA, viruses and monoclonal antibodies. High binding capacity, good product recovery and resolution are also benefits of monoliths. Different samples, (Standard batch purified exosomes, Culture supernatant filtered, Culture supernatant non-filtered), derived from MeWo and LNCap culture media,, were screened. QA, SO3, DEAE and OH CIM 1mL tube - 6μm pore size were screened. CIM® QA - 6μm pores was chosen.

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One of the major requirements for pharmaceutical-grade pDNA is its high homogeneity, being mostly in supercoiled (sc) isoform. Chromatographic separation of sc pDNA from open coiled (oc) or linear isoform is challenging due to their similar interactions with the chromatographic phases. Promising separation efficiency of pDNA isoforms was proven on recently developed histamine modified monolithic chromatographic column in descending ammonium sulfate gradient. The aim of the study was to further optimise the chromatographic conditions for sample analysis, where all three isoforms would be baseline separated.

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Phosphoproteomics is a branch of proteomics that focuses on deriving a comprehensive view of the extent & dynamics of protein phosphorylation by way of identifying & characterizing proteins that contain a phosphate group as a posttranslational modification. One of the approaches for specific enrichment of phosphopeptides from complex samples is metal oxide affinity chromatography (MOAC), where the specific adsorption results from bridging bidentate bindings formed between the phosphate anions and the surface of a metal oxide, such as TiO2, ZrO2, Fe2O3, and Al2O3. In presented study, a rutile TiO2 nanoparticles were bound to a previously polymerised CIM hydroxy monoliths.

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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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