Projects / Programmes source: ARIS

Optimization of methacrylate monolithic supports for macromolecule separation

Research activity

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
Evaluation (rules)
source: COBISS
Researchers (18)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  29065  Matevž Avbelj  Biotechnology  Technical associate  2007 - 2008 
2.  22356  PhD Katja Benčina  Chemistry  Junior researcher  2006 - 2008  53 
3.  14360  PhD Mojca Benčina  Biotechnology  Researcher  2006 - 2008  385 
4.  17915  PhD Helena Gradišar  Biotechnology  Researcher  2006 - 2008  126 
5.  18511  PhD Polona Jamnik  Biotechnology  Researcher  2006 - 2008  900 
6.  17278  MSc Janez Jančar  Chemical engineering  Technical associate  2006 - 2008  61 
7.  24377  PhD Nika Lendero Krajnc  Chemistry  Junior researcher  2006 - 2008  96 
8.  20412  PhD Mojca Merhar  Pharmacy  Researcher  2006 - 2008  33 
9.  17280  Darija Oven    Technical associate  2006 - 2008 
10.  16327  PhD Matjaž Peterka  Biotechnology  Researcher  2006 - 2008  218 
11.  12728  PhD Aleš Podgornik  Chemical engineering  Head  2006 - 2008  706 
12.  08589  PhD Rudolf Podgornik  Physics  Researcher  2006 - 2008  703 
13.  04001  PhD Peter Raspor  Microbiology and immunology  Researcher  2006 - 2008  1,890 
14.  26266  PhD Vida Smrekar  Biochemistry and molecular biology  Researcher  2006 - 2008  35 
15.  17281  Irena Škraba    Technical associate  2006 - 2008 
16.  00779  PhD Aleš Štrancar  Chemical engineering  Researcher  2006 - 2008  479 
17.  19626  Rok Štravs  Biotechnology  Researcher  2006 - 2008  18 
18.  23594  PhD Jana Vidič  Materials science and technology  Junior researcher  2006 - 2008  65 
Organisations (5)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  21,257 
2.  0158  BIA, Laboratory and process equipment company, Ltd.  Ljubljana  5327601  18 
3.  0481  University of Ljubljana, Biotechnical Faculty  Ljubljana  1626914  65,726 
4.  1554  University of Ljubljana, Faculty of Mathematics and Physics  Ljubljana  1627007  33,675 
5.  1655  Sartorius BIA Separations L.t.d., Separations technology company  Ajdovščina  1319612  1,179 
      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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