Tsoka, Sophia; (1996) Real-time monitoring for the enhancement of virus-like particle recovery from yeast. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Virus-like particles (VLPs) are multimeric proteins of particulate nature that are providing the basis for many biological products, including vaccines and diagnostic agents. The efficient production of VLPs depends on the ability to monitor their concentration during recovery. Optical immunochemical monitoring techniques can aid rapid product quantification during process development with their high sensitivity, specificity and speed of measurement. The antibody-VLP interaction leads to specific VLP recognition and has been studied with respect to its use in real-time monitoring of VLPs by dynamic light scattering and with an optical biosensor. The use of dynamic light scattering (DLS) for the rapid evaluation of the concentration and purity of the VLPs in yeast homogenate was investigated. The principle for the development of a DLS-based optical immunoassay involved a change in the signal proportional to the VLP concentration by the addition of antibodies that bind on the VLP surface and detection of that size change by DLS. Changing the ratio of VLPs to cellular particulates led to the calibration of the DLS assay and proved the suitability of the assay for the quantification of VLPs in partially clarified homogenate. The Fisons Applied Sensor Technology (FAST) IAsys optical biosensor that utilised the sensing principle of the resonant mirror was also used to monitor the interaction between VLP and an anti-VLP antibody. The conditions for the optimal quantification of VLPs were identified. Both optical methods were found to provide a significant improvement on rapid monitoring alternatives for VLPs, exhibiting good sensitivity and speed of measurement. Borax flocculation of cell debris followed by PEG precipitation of VLPs was proposed as an initial clarification alternative prior to high resolution purification. The process from fermentation to precipitation as well as the precipitation profile were characterised. Treatment of VLP homogenate with polyethylene glycol (PEG) has been shown to precipitate VLPs selectively, away from contaminating proteins and cellular debris. Real-time monitoring of the PEG precipitation stage using DLS and the optical biosensor was attempted and was proven to be a difficult target. The thesis challenges the suitable assay methods for monitoring proteins such as VLPs in complex process streams. For DLS, the assay was shown to be suitable for monitoring during the fermentation and early purification stages of a downstream process. For the optical biosensor, the final stages of purification (e.g. chromatography) were suggested as the most promising monitoring applications. Manipulation of the affinity support system was recommended to enhance the antibody to VLP interaction in preference to that of non-specific binding on the biosensor surface.

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