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Title: Protein aggregation behaviour and the second virial coefficient
Author: Quigley, Amanda Leila
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2013
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One key barrier to the timely and efficient production of biopharmaceuticals is that they are prone to both chemical and physical instability including aggregation. Protein-protein molecular interactions are known to be a factor in protein solution aggregation behaviour; however their practical and elementary importance has not been fully established. The osmotic second virial coefficient (B22) is a fundamental physiochemical property that describes the molecular interactions between proteins in solution which could result in aggregation. This experimental study reports on the B22 of five different proteins (lysozyme, lactoferrin, catalase, concanavalin A and anti-TNFα dAb) using Self-Interaction Chromatography (SIC) over a wide range of solution conditions including the effects of pH, salt concentration, salt type as well as excipient stabilisers. It was established that current practise for SIC peak analysis was inadequate, and an improved method of SIC peak analysis was deployed which provided improved B22 data quality and robustness. Protein aggregation performance was evaluated under identical solution conditions to those used for the B22 determinations using a simple Dynamic Light Scattering based measurement of initial solution aggregate size. This data shows a direct and strong correlation between B22 values and protein aggregation performance for all systems studied here. Specifically, proteins solution systems with B22 values of ~ 1 x 10-4 mL mol g-2 or less, regardless of solution composition, all exhibited aggregation behaviour. This thesis establishes the decisive importance of B22 as a predictor of aggregation performance. Preliminary validation data is presented for a method which allows for B22 estimation from osmotic cross virial coefficients (B23), removing the need for immobilisation of the protein of interest and therefore allowing rapid B22 screening.
Supervisor: Williams, Daryl ; Heng, Jerry Sponsor: FUJIFILM Diosynth Biotechnologies
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available