Burden, CS; (2013) Monolith absorbants as a capture step for virus-like particles. Doctoral thesis (PhD), UCL (University College London).

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Abstract

Monoliths are an alternative stationary phase format to conventional particle based media for large biomolecules. Conventional resins suffer from limited capacities and flow rates when used for viruses, virus-like particles (VLP) and other nanoplex materials. Monoliths provide an open pore structure to improve pressure drops and mass transport via convective flow. The challenging capture of a VLP from clarified yeast homogenate was used to develop a new monolith separation which found hydrophobic interaction based separation using a hydroxyl derivatised monolith had the best performance. The monolith was then compared to a known beaded resin method, where the dynamic binding capacity increased three-fold for the monolith with 90% recovery of the VLP. Confocal microscopy was used to visualise lipid contaminants, deriving from the homogenised yeast. The lipid formed a layer on top of the column, even after column regeneration, resulting in increasing pressure drops over a number of cycles. Removal of 70% of the lipid pre-column by Amberlite/XAD-4 beads significantly reduced the fouling process. Applying a reduced lipid feed versus an untreated feed further increased the dynamic binding capacity of the monolith from 0.11 mg/mL column to 0.25mg/mL column. Control of chromatographic conditions can impact the product concentration during elution. Critical parameters which influenced the concentration of measureable VLP eluted included column contact time, salt concentration in mobile phase, and inclusion of lipid. The parameters were co-dependant with a crude lipid feed loaded at low salt and extra wash time of 40 minutes causing the largest decrease of 40%. Reducing the time of contact between the column and the VLP helped reduce such adverse effects. Increasing the flow rate in the column had no effect on the elution profile with crude or reduced lipid feeds. This informs process development strategies for the future use of monoliths in vaccine bioprocessing.

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