Projects / Programmes
Optimization of methacrylate monolithic supports for macromolecule separation
| Code |
Science |
Field |
Subfield |
| 2.02.00 |
Engineering sciences and technologies |
Chemical engineering |
|
| Code |
Science |
Field |
| T390 |
Technological sciences |
Polymer technology, biopolymers |
monolithic supports, macromolecules purification, DNA, viruses, polymerization, interactions, ligand synthesis
Researchers (18)
Organisations (5)
Abstract
Chromatographic monoliths are one of the most studied chromatographic stationary phases in recent years. The reason is in very simple preparation especially preparation of capillary columns and microchips, high porosity resulting in low pressure drop, convection transport of molecules that enables very short separation times and flow –unaffected properties like resolution and dynamic binding capacity and finally very high capacity for large macromolecules like DNA and viruses.
During the project we are going to study monolithic structure and its impact on hydrodynamic and chromatographic properties such as capacity, recovery and molecules stability. Optimization of the structure will be performed by changing composition of polymerization mixture or polymerization conditions.
Together with structure optimisation, optimization of mobile phase will be performed too. In this part interaction of macromolecules will be intensively studied with the aim to understand better mechanism of separation.
Significant number of experiments will be dedicated to introduce different types of active groups on the pore surface. Active groups might consist of branched ligands like dendrimers, with the aim to increase binding capacity for differently size molecules, pseudoaffinity ligand useful for purification of recombinant proteins and affinity ligands where particularly effect of the spacer type will be studied. This work will be performed with the goal to increase efficiency of the immobilization.
Significance for science
Theoretical investigations indicates that monoliths should have much higher capacity in comparison to particle shape supports and are therefore very promising stationary phase for purification of large proteins, DNA or even viruses. One of the main problems during loading of large amounts of such molecules is that they might irreversibly bind to the matrix as a result of strong interaction between the molecule and the matrix, and that they are sensible to degradation. We demonstrated that indeed there is partial degradation of extremely large molecules like large plasmids or genomic DNA but that it is possible to purify under optimal conditions even plasmids up to 90 kbp preserving most of its supercoiled form. We also demonstrated that there is substantial increase in plasmid binding capacity with addition of NaCl to the loading solution. This is attributed to neutralisation of negative charges between DNA molecules bound to the matrix what results in entropic driven binding process. This influences also recovery affected by partial irreversible binding to the matrix. Another important achievement demonstration that grafting of the monolith doubles binding capacity even for very large molecules like plasmid DNA. It was shown that it is possible to purify bacteriophages in a way that preserve their viability.
Significance for the country
Company BIA Separations d.o.o. already commercialize monolithic chromatographic columns. Results of the project are procedures for preparation of large monolithic plates, determination of optimal monolith structure for purification of extremely large molecules like plasmid DNA or viruses, procedure for preparation of grafted monoliths and development of non-invasive method for determination of ligand density measuring pH transient. These results increase competitive advantage of monolithic supports over other media and consequently increase sell and market share, resulting in increase of number of employees. In addition, this increase worldwide visibility and classifies Slovenia as a high tech country.
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Most important socioeconomically and culturally relevant results
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