The experiments documented in this thesis investigate the use of aqueous two-phase systems (ATPS) as a viable method for the processing of influenza virus particles in a feedstock derived from embryonated hen's eggs. The virus particles are currently purified in an industrial process using stages of sucrose density gradient ultracentrifugation that produce a subunit vaccine Fluvirin which consists of the immunoprotective antigens haemagglutinin (HA) and neuraminidase (NA). The current purification scheme suffers process bottlenecks in particular, limitations of scale hence volumetric throughput and time taken to produce a batch that could be circumvented by ATPS. Manipulations of polyethylene glycol (PEG) molecular weight, tie-line length (TLL) and volume ratio were exploited for the design of aqueous two-phase systems. A simple two-stage process was designed in which, (i) 60 % (by mass) of contaminating proteins were eliminated in an ATPS comprising PEG 300 22.2 % w/w/ phosphate 17.9 % w/w at pH 7.5 and (ii) HA antigen was released from intact influenza particles in the presence of 1 % w/w Triton X100 in an optimised secondary ATPS comprising PEG 300 13.9 % w/w/ phosphate 24.3 % w/w, pH 7.5. The first purification stage using ATPS performed as well as the ultracentrifugation method. The material was produced with purification factor of 4.2 and intact virus recovery of 72 % within one hour, (as compared to a purification factor 4.0 and recovery of 109 % within 18 hours, using ultracentrifugation). Thus ATPS has demonstrated potential in fractionating particulate feedstocks in relatively short times, thus facilitating the circumvention of bottlnecks in the current commercial process.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:396445 |
| Date | January 2002 |
| Creators | Aiyedebinu, Adetoun Olufolake |
| Publisher | University of Birmingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://etheses.bham.ac.uk//id/eprint/4177/ |
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