An investigation of the properties of bacteriophage
M13 and the implications for its large-scale
Doctoral thesis, UCL (University College London).
Bacteriophage are a diverse class of viruses that infect bacterial cells. As a result of over 60 years of molecular biology advances, bacteriophage today feature as candidates for vaccination, gene therapy, biomaterial and antibacterial purposes. Consequently, scientific, commercial and public awareness of bacteriophage is growing rapidly. There is now an increasing need for the establishment of strong biochemical engineering foundations to serve as a guide for future bacteriophage bioprocessing. It has been the purpose of this study to contribute towards this knowledge base, by understanding the properties of the filamentous bacteriophage M13. Ultimately, this work has aimed to allow for the more efficient assembly of a large-scale production process. By the application of well-understood small-scale predictive techniques, it has been found that bacteriophage M13 should not be severely damaged by hydrodynamic shear forces of the duration and magnitude imparted by fermentation, pumping or continuous centrifugation operations. Thus, it may well be possible to manufacture on the large-scale using existing large-scale equipment designs. Amongst bacteriophage, the reproduction strategy of M13 is unusual in that propagation occurs by the non-lethal extrusion of progeny through the cell wall of the E. coli host. Investigation of bacteriophage M13 propagation indicated that growth in a medium that increased host cell density concomitantly increased bacteriophage yield; a four-fold increase to 2 x 1012 pfu ml-1 was achieved. At the end of culture, concentrations of supernatant DNA and protein contaminants were found to vary amongst three E. coli strains studied. Post-fermentation, bacteriophage M13 can be precipitated from the cell-free process fluid by as little as 2 % (w/v) PEG 6 000 plus 25 mM magnesium sulphate, or by isoelectric precipitation. Purification factors in excess of 100 were achieved by PEG-salt precipitations with regards to the reductions in DNA and protein concentrations. Methods used in this study have increased the processing knowledge of bacteriophage M13 and have a broader applicability to both derivatives of M13 and other bacteriophage.
|Title:||An investigation of the properties of bacteriophage M13 and the implications for its large-scale bioprocessing|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of BEAMS > Faculty of Engineering Science > Biochemical Engineering|
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