Abstract
BackgroundPlasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression. The increasing number of clinical trials on plasmid-based measles vaccines has triggered the need to make more in less time.ResultsIn this work, we investigated the process variables necessary to improve the volumetric and specific yields of a model plasmid-based measles vaccine (pcDNA3F) harboured in E. coli DH5α. Results from growth medium optimisation in 500 mL shake flasks by response surface methodology (RSM) generated a maximum volumetric yield of 13.65 mg/L which was 1.75 folds higher than that of the base medium. A controlled fed-batch fermentation employing strategic glycerol feeding and optimised growth conditions resulted in a remarkable pcDNA3F volumetric yield of 110 mg/L and a specific yield of 14 mg/g. In addition, growth pH modification and temperature fluctuation between 35 and 45°C were successfully employed to improve plasmid production.ConclusionProduction of a high copy number plasmid DNA containing a foreign gene of interest is often hampered by the low plasmid volumetric yield which results from the over expression of foreign proteins and metabolic repressors. In this work, a simple bioprocess framework was employed and successfully improved the production of pcDNA3F.
Highlights
Plasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression
Prior to the medium optimisation, a preliminary medium screening based on Plackett Burman design [21,22] was carried out to select three most significant medium components in plasmid DNA semi-defined medium (PDM) and the results showed that tryptone, glucose and disodium hydrogen orthophosphate (Na2HPO4) significantly affected pcDNA3F volumetric yield
It has been observed that the inclusion of fusion protein gene (F) of measles virus into base plasmid pcDNA3 resulted in reduced plasmid volumetric yields during fermentation compared to naked pcDNA3
Summary
Plasmid-based measles vaccines offer great promises over the conventional fertilised egg method such as ease of manufacture and mimic wild-type intracellular antigen expression. The increasing number of clinical trials on plasmid-based measles vaccines has triggered the need to make more in less time. The enhanced thermal stability of plasmid DNA at room temperature and above offers a great promise for the treatment of measles and other diseases in tropical and economically disadvantaged areas [3]. The duration at which the cells are exposed to a certain temperature shift is known to affect the maximum recombinant protein production [12,13]. It is further suggested that a cooling step may be required to drive cells to maximum potential plasmid copy number which may not be possible at higher temperatures [16]
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