Development of monolithic chromatographic columns with small volumes (in continuation: mini discs) in presented in this master's thesis. A 3-part chromatographic housing made from polyoxymethylen copolimer (POM-C) was developed. It enables the usage of three different size-classes of mini discs (50µL, 100 µL and 200 µL), which can be used either on HPLC or FPLC systems, or manually via the attachment on syringe. The optimization of housing was achieved by reducing its dead volume and non-specific binding adsorption of bio-molecules on chromatographic frits. Demonstration of successful preparation of affinity mini disc was achieved by immobilization of a recombinant protein A on stationary phase (monolith). With the implementation of preliminary qualitative characterization of mini discs in the initial development phase – hydroxy modification, such as pulse response (integrity test) and benzimidazole dynamic binding capacity as analog of IgG (test of homogeneity), we achieved optimization of immobilization and the entire process of monolith affinity chromatography preparation. Preparation of immobilized enzyme reactor was achieved by covalent immobilization of TEV protease that was previously isolated from Escherichia coli. The immobilization was performed on various activated mini discs carriers (carbonyl diimidazole (CDI) and aldehyde groups). Activity of TEV protease was examined by cleaving the affinity histidine tag from recombinant proteins Listeriolysine O and DARPin. The cleavage of his-tag on mini discs IMERs with TEV protease was not successful, in spite of successful cleavage of His- tagged DARPin obtained by TEV protease in solution. In order to achieve successful cleavage, further optimization of is required (covalent binding using an aldehyde spacer, or disulfide covalent binding via thiol groups of cysteine residues), as well as stability and activity testing of the enzyme in various immobilization buffer solutions.
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