Bláha, Benjamin A. F.; (2018) Platform processes for vaccine production: Development of a universal influenza vaccine using Pichia pastoris. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Influenza virus remains a familiar threat to public health and continues to challenge the scientific community. Most challenging is responding to the evolutionary adaptability of influenza virus, which often hinders effective prevention or treatment. Furthermore, current production methods are logistically and technically inadequate to cope with pandemic surges, leaving a considerable number of individuals unprotected. Tandem Core Virus-Like Particles (VLPs), expressed in Pichia pastoris, offer an exciting proposition to create a platform process for a universal influenza vaccine. However, as with all novel concepts, characterisation of this technology was required and process methodologies were developed accordingly. Initially, critical process parameters, associated with production of novel VLP constructs, were identified. In doing so, a need for a robust, high-throughput miniaturised fermentation platform was recognised. To accommodate such an approach, a high-throughput, non-contact, automated, small-scale, scalable disruption tool was developed to extract VLPs from P. pastoris. Development and optimisation of this method led to matching and even outperforming High Pressure Homogenisation performance. Having developed the prerequisite tools for miniaturised upstream process development, the use of microtitre plates for studying heterologous protein expression was investigated. This proved to be challenging, predominantly due to reduced mass transfer capacity imposed by microtitre plates and the requirement of controlled methanol feeding. The use of pectin digest as an alternative indirect induction agent was studied. It was found that the use of this novel media resulted in the expression of heterologous protein, however, this effect was most likely a result of methanol liberation during digest preparation. Due to the requirements of a tightly controlled induction process, it was found that, microtitre plates provided an unsuitable platform for rapid upstream process development for methylotrophic yeast. As an alternative to microtitre plates, the effects of variance of previously identified critical process parameters were studied to a greater extent using miniaturised bioreactors, initially with a simplified, non-epitope-exposing, variant of Tandem Core VLP. Metabolic responses, such as final biomass concentration, required significantly different optimum operating conditions than product expression responses. It was also found that maximising expression of heterologous protein was not synonymous to maximising the production of VLPs. After characterising VLP expression in miniaturised bioreactors, these findings were translated to industrially relevant universal influenza vaccine production scenarios. This was accomplished by investigating the effects of scale-up and variation of epitope inserts that could be utilised as a universal influenza antigens. Following this, the scale-up of fermentation processes and was studied. It was found that mixed induction with glycerol feeding not only provided a better basis for scale-up but also resulted in higher product titres for more complex VLP constructs.

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