With the development of regenerative medicine and tissue engineering, the
need to develop affinity chromatographic methods has increased. The bottleneck of
chromatography, however, still remains the purification and isolation of large quantities
of specific biologic mamcromolecules and nanoassemblies, including certain
populations of cells, such as stem cells. Convective affinity chromatography is a very
effective method for separating large biomolecules such as larger proteins, viruses,
bacteriophages, DNA and cells. Monoliths have a characteristic porous structure where
the pores are interconnected and thus allow the rapid exchange of molecules on the
basis of convective mass transport. A very important challange, particularly connected
to large macromolecules and cells, are non-specific interactions. Frequently this issue is
solved by introduction of a hydrophilic layer. A common approach is the introduction of
polyethylene glycol (PEG), however, for affinity chromatography, a better option is
dextran with a higher number of reactive moieties, resulting in higher density of
biologically active molecules. The introduction of dextrane layers was the main goal of
this master thesis.
The master thesis can be divided into three sections. In the first part I successfully
prepared small volume porous polymer supports. This was followed by the chemical
modification with ethylenediamine and carbonyldiimidazole. In the second set I focused
on the development of dextrane layers, that will improve the covalent immobilization of
biomolecules. I oxidized the dextran with the sodium periodate according to the
principle of the Malaprad reaction and bound it covalently to the developed
chromatographic columns. For the purpose of evaluating oxidized dextran, I
implemented quantitative BCA methods for the determination of carbonyl groups,
originally proposed for determination of proteins. In the last set of experiments, I
prepared CDI and epoxy porous polymer supports on which I immobilized antibodies to
the CD20 antigen and incubated them in the blood lysate of a patient with KLL. The
lymphocytes bound to the porous polymer supports were observed by light and
fluorescence confocal microscopy.
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