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Projects / Programmes source: ARIS

Innovative production systems for vaccines and regenerative medicine

Research activity

Code Science Field Subfield
4.06.00  Biotechnical sciences  Biotechnology   

Code Science Field
T490  Technological sciences  Biotechnology 

Code Science Field
2.09  Engineering and Technology  Industrial biotechnology 
Keywords
purification, cells, VLPs, bacteriphages, supports, monoliths, chromatography
Evaluation (rules)
source: COBISS
Researchers (20)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  15627  PhD Ariana Barlič  Biochemistry and molecular biology  Researcher  2011 - 2014  89 
2.  12667  PhD Miloš Barut  Chemistry  Researcher  2011 - 2014  206 
3.  29874  Tina Brne    Technical associate  2011 - 2012 
4.  19029  PhD Nina Daneu  Materials science and technology  Researcher  2011 - 2014  420 
5.  30197  PhD Lenart Girandon  Biotechnology  Researcher  2011  40 
6.  27827  PhD Jon Gutierrez Aguirre  Biotechnology  Researcher  2011 - 2014  361 
7.  33406  PhD Nikolaja Janež  Biochemistry and molecular biology  Researcher  2011 - 2014  81 
8.  24078  Sandra Kontrec    Technical associate  2011 - 2014 
9.  16002  PhD Nevenka Kregar Velikonja  Biotechnology  Researcher  2011 - 2014  317 
10.  35240  Miroslava Legiša    Technical associate  2013 - 2014 
11.  24377  PhD Nika Lendero Krajnc  Chemistry  Researcher  2011 - 2014  96 
12.  29178  PhD Dany Morisset  Biotechnology  Researcher  2011 - 2013  121 
13.  16327  PhD Matjaž Peterka  Biotechnology  Researcher  2011 - 2014  218 
14.  12728  PhD Aleš Podgornik  Chemical engineering  Head  2011 - 2014  709 
15.  05229  PhD Maja Ravnikar  Biotechnology  Researcher  2011 - 2014  1,360 
16.  10083  PhD Aleksander Rečnik  Chemistry  Researcher  2011 - 2014  651 
17.  26266  PhD Vida Smrekar  Biochemistry and molecular biology  Researcher  2011  35 
18.  29635  Katja Stare  Biology  Researcher  2011  86 
19.  00779  PhD Aleš Štrancar  Chemical engineering  Researcher  2011 - 2014  479 
20.  09864  PhD Magda Tušek Žnidarič  Biology  Researcher  2012 - 2014  412 
Organisations (5)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0105  National Institute of Biology  Ljubljana  5055784  13,315 
2.  0106  Jožef Stefan Institute  Ljubljana  5051606000  89,989 
3.  1655  Sartorius BIA Separations L.t.d., Separations technology company  Ajdovščina  1319612  1,189 
4.  3030  Center of Excellence for Biosensors, Instrumentation and process Control  Ajdovščina  3660460  939 
5.  7421  EDUCELL cell therapy service Ltd. Ljubljana  Trzin  1198327  643 
Abstract
Over the past decade there has been a rapid development in different pharmaceutical and biotechnological fields. Biological drugs, based especially on monoclonal antibodies, are now more and more often used in treatment of heavy diseases. Also many other ways of treatment are being investigated, especially on fields of vaccines, antibiotic resistance, gene and cell therapy and regenerative medicine. Virus like particles (VLP) are intensively investigated for their potential use in vaccination. A little less examined in this field are bacteriophages, which are on the other hand extremely interesting as an antibiotic alternative. Besides, stem cell research is now in increase for potential use in tissue engineering, cell therapies and regenerative medicine. While isolation of VLP's, bacteriophages and cells on laboratory scale is not problematic, there are several challenges when large amounts of products with high purity have to be prepared. Today, there are two mostly used methods for separation and purification of stem cells: flow cytometry (FACS) and immunomagnetic separation (MACS), however, both are too expensive to be used on industrial scale. To achieve efficient cultivation of VLP’s and bacteriophages choosing of an appropriate expression system is very most important but also novel effective methods for purification on industrial scale should be developed. Traditional methods, such as filtration and centrifugation, allow production of large amounts, but required purity is hardly achieved. This is the main reason why chromatography is lately intensively investigated for purification of VLP’s and bacteriophages. Appropriate robust cell line will be used for testing. VLP’s will be propagated in plants with the goal to achieve sufficient quantity. Norovirus particles have been chosen, because noravirus is the major cause of non-bacterial gastroenteritis. Selected bacteriophages have also been chosen because of their potential use. Targets are bacteria from genus Campylobacter, which cause between 400 and 500 million food infections per year, and from genus Erwinia and Acidovorax, which cause great loss in plant food and decorative plant production. We will also optimize phage cultivation conditions, because they have significant effect on the productivity. We will define appropriate bacterial system, optimize cultivation parameters in bioreactors, as well as determine appropriate time for infection and termination of the bioprocess. The main part of the research will be development of suitable convective based supports, which will allow efficient chromatographic purification of cells, VLP’s and bacteriophage. One approach will be optimization of existing methacrylate monoliths, especially in terms of pore diameter and type of ligand. Length of grafted chains and their density will be optimized to achieve high permeability, dynamic binding capacity and yield. We will also try to develop fractal chromatographic support, which should theoretically have even better characteristics than existing ones. For cell purification a new generation of support will be develop, based on inorganic porous support, hollow fibres and macroporous chromatographic monoliths. In parallel, we will be test different chemical modification of pore surface, since it is known that the biggest problem in cell separation and purification, is their desorption. To overcome this problem, we will develop different types of ligands, which will either be cleaved at different mobile phase compositions or will change their configuration. These are so called thermoresponsive ligands, which could at temperature changes of the mobile phase cause cell desorption. Novel  convective supports  prepared during this project  will be tested on cells, VLP’s of norovirus and various bacteriophage systems. In second part of the project we will further optimize developed supports and methods with a goal to achieve highly efficient separation and purification of target pro
Significance for science
Project results contributed to better understanding of adsorption phenomena mechanisms, characterization of convective monolithic chromatographic supports and also leaded to development of novel functionalization procedures. Grafted methacrylate monoliths bearing different functional groups on the same support enable study of complex adsorption phenomena affected by surface diffusion, countercurrent diffusion, entropic stabilization of low molecular compound inside grafted layers and steric exclusion, again as a consequence of grafted layer. We also demonstrated that that inside grafted layers activity of functional groups differs from the activity of same type of groups linked directly on monolith skeleton. Based on this finding it is possible to better describe adsorption and consequently simpler and more efficient incorporation of such supports into downstream processing. In the field of inorganic supports experiments performed during this experiment demonstrated possibility of rutile twin hydrothermal synthesis what might result in novel type of convective supports applicable also to downstream processing but also in many other areas like e.g. catalysis. We also demonstrated that it is possible to produce bacteriophages in continuous processes together with the fact that by optimization of growth conditions we achieved 2-fold higher maximal specific growth of Campylobacter that previously described in the literature. Another interesting conclusion is related to norovirus VLPs for which it was shown that they are not degraded by shear forces caused by HPLC.
Significance for the country
Developed methods based on monolithic chromatographic supports enable significantly faster and more efficient production of bacteriophages and VLPs with purity suitable of human treatment. This is of extreme importance in case of epidemic or even pandemic threat where each day of delay in providing vaccines might results in several hundred or even thousands of casualties. As such processes provide for an order of magnitude higher productivity it is obvious sufficient quantities can be produced. Due to lower number of required purification steps the overall production costs are substantially reduced enabling much lower costs of final products based on bacteriophages and VLPs that might be used as antibiotic substitutes or vaccines, and therefore much broader accessibility for humans worldwide. Because of that it can be anticipated that result produced throughout this project might have substantial impact on human health in general wile on the other hand represent substantial added values for investor increasing accessibility to new markets and consequently need for new employments.
Most important scientific results Annual report 2011, 2012, 2013, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2011, 2012, 2013, final report, complete report on dLib.si
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